St - B17 serotonin receptor

ABSTRACT

Genes encoding the St-B17 serotonin receptor protein were cloned and characterized from a rat striatum mRNA and a human genomic library. The St-B17 receptor has nucleotide and amino acid homology with previously described 5-HT genes and can bind ligands that are known to interact with serotonin receptors. In addition, the levels of intracellular cAMP in cells transfected with the receptor gene respond in a dose dependent manner to introduction of serotonin in the media.

SCOPE OF THE INVENTION

[0001] This invention relates to cloning and characterization ofcellular receptors. Specifically, this invention relates to the cloningand characterization of the St-B17 serotoiin receptor protein.

BACKGROUND

[0002] The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has avariety of functions in the central nervous system. It has beenimplicated in many cognitive and behavioral functions, includingaggression, sexual behavior, learning and sleep. Disruptions ofserotonergic systems may be a critical factor in a number of clinicaldisorders or conditions including schizophrenia, depression, obsessivecompulsive disorder, anxiety, migraine headaches, and pain.

[0003] The multitude of effects produced by serotonin are mediated byvarious serotonin receptors which exist in the central and peripheralnervous system. The transduction of serotonergic signals across theneuronal membrane is mediated by a diversity of receptor subtypes which,in mammals, appear to fall into four pharmacologically distinct classesdesignated 5-HT₁-5-HT₄. The 5HT₁ subcategory has been further subdividedinto five different subtypes referred to as 5HT_(1A-E). The primarystructures for a number of these receptors have been elucidated bymolecular cloning, including the 5-HT₁, 5-HT₂ and 5HT₃ subclasses. Inaddition, the sequences of three different Drosophila serotoninreceptors, 5-HT_(dro1) and 5-HT_(dro2A,B), have been reported.

[0004] Selective therapeutic agents, including agonist and antagonistdrugs, have been developed based on serotonin receptor technologies.5-HT₂ antagonists, for example, are useful in the treatment ofschizophrenia, parkinsonism, and anxiety disorders. Several azapirones,such as buspirone, gepirone, and ipsapirone, have high affinities for5HT_(1A) receptors in the brain, and are useful in the treatment ofanxiety. Highly selective 5-HT uptake inhibitors, which have minimaleffects on norepinephrine or dopamine uptake or on otherneurotransmitter receptors, have been used to successfully treatdepression.

[0005] Characterization of all specific 5-HT receptors would clarify therole of serotonin in the central nervous system. Analysis of thereceptor subtypes and their functional role in the central nervoussystem would help elucidate the pathophysiological basis of many humandiseases. Accordingly, there is a need for identification andcharacterization of all specific 5-HT receptors, and the development ofselective therapeutic agents based on these receptor technologies.

DESCRIPTION OF THE FIGURES

[0006]FIG. 1: Saturation analysis of specified [¹²⁵I]-LSD binding tomembranes prepared from Cos-7 cells transiently transfected withconstruct pSRα-B17. The experiment shown is representative of 3independent experiments conducted in triplicate. Inset, scatchardanalysis of saturation binding data. In this experiment the K_(D) andB_(max) values were 1.5 nM and 3.4 pMoles mg protein⁻¹, respectively.

[0007]FIG. 2: Pharmacological analysis of [¹²⁵I]-LSD binding to Cos-7cell membranes expressing clone St-B17. Competition curves shown arerepresentative of 3 independent experiments conducted in triplicate.

[0008]FIG. 3: Northern blot analysis of St-B17 RNA transcripts in ratbrain. Each lane contained 3 μg of poly (A+) RNA. Lanes: AMYG, amygdala;CB, cerebellum; CTX, cerebral cortex; HIP, hippocampus; HYP,hypothalamus; MED, medulla; OB, olfactory bulb; OT, olfactory tubercle;PIT, pituitary; RET, retina; STR, striatum; THAL, thalamus. Locations ofRNA size (kb) markers are indicated.

[0009]FIG. 4: Accumulation of cAMP in response to agonist activation ofSt-B17 stably expressed in HEK-293 cells. This figure shows adose-response curve activation of St-B17 by serotonin in stablytransfected, intact HEK-293 cells. FIG. 5: Pharmacological analysis ofserotonin stimulated cAMP accumulation in stably transfected HEK-293cells. DE is an acronym for dihydroergocryptine. All drugs were assayedat 10 μM final concentration.

SUMMARY

[0010] One embodiment of the present invention is the isolated mammalianserotonin receptor protein St-B17. Preferably this receptor protein ishuman. The present invention also encompasses species variations of theSt-B17 receptor.

[0011] Another embodiment of the present invention is a method forscreening a drug candidate for central nervous system activity bycontacting the drug candidate with the St-B17 protein and measuringbinding of the drug candidate by the protein.

[0012] An additional embodiment of the present invention is a method forscreening a drug candidate for central nervous system activity by firstcontacting the St-B17 serotonin receptor protein with a first moleculeknown to be bound by the protein to form a first complex of the proteinand the first molecule; then contacting the first complex with the drugcandidate; and finally measuring whether the drug candidate displacesthe first molecule from the first complex. This method can preferablyinclude in the measuring step, the step of measuring the formation of asecond complex of the protein and the drug candidate. An alternativemethod of measuring the displacement of the first molecule can beperformed by measuring the amount of the first molecule that is notbound to the protein.

[0013] A further embodiment of the present invention includes arecombinant construct of the polynucleotide, preferably thepolynucleotide of SEQ ID NO: 7, encoding the St-B17 serotonin receptorprotein operably linked to a heterologous promoter.

[0014] Another embodiment of the present invention is directed at thepolynucleotide sequence encoding the St-B17 receptor. Preferably, thispolynucleotide sequence is Sequence ID NO: 7 or species variationsthereof. Additionally, the present invention encompasses an isolatednucleotide sequence having homology to at least 18 contiguousnucleotides of the St-B17 gene.

[0015] The present invention also embodies a mammalian cell line,preferably human, in continuous culture expressing the St-B17 serotoninreceptor protein. Particularly, the cell line contains thepolynucleotide Sequence ID NO: 7 or species variations thereof. Oneespecially preferred embodiment includes HEK 293 cells in continuousculture expressing the St-B17 receptor.

[0016] Yet another embodiment of the present invention includes isolatedantibodies against the St-B17 serotonin receptor protein. Preferablythese isolated antibodies are polyclonal, even more preferably, theisolated antibodies are monoclonal.

[0017] An even further embodiment of the present invention includes amethod of screening a ligand for binding to the St-B17 serotoninreceptor by the steps of:

[0018] transfecting a cell line with a gene encoding the St-B17serotonin receptor protein in an expression vector;

[0019] culturing the cell line to express the gene in media containing aligand of the receptor; and

[0020] measuring the binding of the ligand the receptor produced by thecell line.

[0021] Preferably, in this method, the cell line is from a mammal, morepreferably a human, and most preferably the cell line is HEK 293. Anadditional preferred embodiment of the above method entails transfectingcells with the St-B17 serotonin receptor encoded by the polynucleotideof SEQ ID NO: 7. Additional preferred embodiments of this method includeusing eukaryotic expression vectors, preferably pSRα-B17. Anotherpreferred embodiment includes using ligands that are labeled and thelabel is either radioactive or calorimetric.

[0022] Still another embodiment of the present invention is a method ofdetermining the ability of a drug to inhibit ligand binding to theSt-B17 serotonin receptor protein by the following steps:

[0023] transfecting a cell line with a DNA sequence encoding St-B17 inan expression vector;

[0024] culturing the cell line to express the St-B17 receptor in mediacontaining a ligand of the receptor;

[0025] determining the level of binding of the ligand to the receptor;

[0026] culturing the same cell line to express the receptor in thepresence of both the ligand and the drug; and

[0027] determining the level of binding of the ligand to the expressedreceptor, wherein a lower level of binding in the presence of the drugindicates that the compound is an inhibitor of ligand binding.

[0028] Preferably, the cell line of this method is mammalian, mostpreferably HEK 293, and the ligand, the drug or both are labeled.Additionally, the expression vector of this method can preferably bepSRα-B17 and the ligand can advantageously be serotonin.

[0029] Yet another embodiment of the present invention is a method fordetermining the ability of a drug to inhibit ligand binding to theSt-B17 serotonin receptor by the following steps:

[0030] transfecting a cell line with a gene encoding the St-B17 receptorprotein in an expression vector;

[0031] culturing the cell line to express the receptor;

[0032] determining the level of binding of the ligand to the receptor;

[0033] culturing the same cell line to express the receptor in thepresence of both the ligand and the drug being tested for inhibition;and

[0034] determining the level of cAMP accumulation in the cell, wherein alower level of cAMP accumulation in the cell in the presence of the drugindicates inhibition of ligand binding.

[0035] Still another embodiment of the present invention is a method ofdetermining the ability of a drug to inhibit ligand binding to theSt-B17 serotonin receptor protein by the steps of:

[0036] transfecting a cell line with a DNA sequence encoding St-B17 inan expression vector;

[0037] culturing the cell line to express the St-B17 receptor in mediacontaining a ligand of the receptor;

[0038] isolating the membranes from the cell line, wherein the membranescontain the expressed St-B17 receptor;

[0039] determining the level of binding of the ligand to the receptor onthe membranes;

[0040] incubating the membranes in the presence of both the ligand andthe drug; and

[0041] determining the level of binding of the ligand to the membranes,wherein a lower level of binding in the presence of the drug indicatesthat the compound is an inhibitor of ligand binding.

[0042] Still another embodiment of the present invention is a method fordetecting expression of the St-B17 serotonin receptor gene in a tissuesample by the steps of:

[0043] removing a tissue sample from a mammal;

[0044] contacting the mRNA in the cytoplasm of the tissue sample with alabeled polynucleotide probe having homology to the St-B17 serotoninreceptor gene; and

[0045] detecting the binding of the probe to the tissue wherein positivebinding indicates expression of the St-B17 serotonin receptor gene.

DETAILED DESCRIPTION

[0046] We have cloned a new serotonin 5-HT (St-B17) receptor gene fromrat and human brain cells. This receptor has a nucleotide sequence thatis distinct from any previous gene in the serotonin family. The ratSt-B17 gene has a 1311 bp open reading frame coding for a protein with437 amino acids. The human St-B17 gene has a 1320 bp open reading framecoding for a protein with 440 amino acids.

[0047] In addition, this receptor protein exhibits a distinctpharmacological profile not previously described. The uniquepharmacology together with the relatively low level of homology (<50%)of St-B17 with previously cloned 5-HT receptor subtypes indicates thatthis receptor does not belong to any of the previously defined5-HT₁-5-HT₄ subcategories of 5-HT receptors. Thus, St-B17 represents anunknown and uncharacterized receptor.

[0048] The molecular cloning of St-B17 will be useful for developingdrugs which interact with 5-HT receptors. For instance, the cloning ofthis novel gene allowed us to develop new assays for discoveringserotonin receptor binding and inhibitory drugs. These assays wereperformed by transfecting cells, such as Cos-7 mammalian cells, with thegene encoding the St-B17 serotonin receptor protein in an expressionplasmid. The Cos-7 cells were then placed in media and expressed theSt-B17 receptor on their cell surface. Radiolabeled ligands were thentested for binding to these transfected cells.

[0049] By using this method we were able to screen many drugs for theirbinding activity to the serotonin receptor. Similarly, this assay systemwas used to detect competitive inhibitors of serotonin binding. Byincubating the transfected cells in the presence of a radiolabeled knownbinding molecule (LSD) we were able to test various drugs for theirability to inhibit this binding. Table 1 below describes a suite ofdrugs that had wide-ranging affects on ¹²⁵I-LSD binding to the ratSt-B17 serotonin receptor.

[0050] Since serotonin binding is intimately involved in thefunctionality of the central nervous system, assays for detecting drugswhich bind or block serotonin receptors are of paramount importance. Asdiscussed in the background, many behavioral functions are believed tobe mediated through serotonergic systems. For researchers to be able toaccurately assess the projected effects of a new serotonin-related drugin humans, it is important to test that drug's effect on every knownserotonin receptor. The discovery of the St-B17 receptor provides theresearcher with a previously unknown assay to study possible in vivoeffects of the drug being tested.

[0051] It is also anticipated that isolated St-B17 receptor protein willbe useful for performing assays to determine agents which bind orinhibit binding to serotonin receptors. In discussing the isolatedSt-B17 receptor protein, we are referring to not only affinity-purifiedprotein, but also receptor proteins bound in a membrane and removed fromthe cell. For example, we transfected Cos-7 cells with the gene encodingthe St-B17 receptor protein in an expression plasmid. After incubationin media to allow St-B17 receptor expression the cells were lysed andthe membranes (containing the bound St-B17 receptors) were pelleted.These membranes, isolated from the host cells were used for radiolabeledligand binding studies.

[0052] The present invention includes isolated St-B17 serotoninreceptors from rats, humans, other mammals, and other vertebrates.Further investigations of St-B17's role in vivo and in vitro will helpimprove current therapies for several neuropsychiatric disorders byproviding additional information on each drug's receptor-mediatedaction.

[0053] For the first step in cloning the St-B17 receptor we used thepolymerase chain reaction (PCR) to selectively amplify cDNA sequencesfrom mRNA purified from rat corpus striatum. Poly(A)⁺ RNA from the ratstriatum was used to first synthesize a cDNA library by reversetranscription using methods well known in the art. The following exampleshows one method for amplifying the serotonin receptor genes using PCRamplification.

EXAMPLE 1

[0054] PCR Amplification of Rat Striatum mRNA

[0055] We used a pair of highly degenerate primers, with sequences whichwere derived from the third and sixth transmembrane (TM) regions ofpreviously cloned G protein-linked receptors in multiple rounds of PCRto amplify the serotonin gene sequences in the striatum library.

[0056] Polymerase chain reaction amplification was carried out aspreviously described (Monsma, F. J., Jr., Mahan, L. C., McVittie, L. D.,Gerfen, C. R. & Sibley, D. R., Proc. Natl. Acad Sci. USA 87, 6723-7(1990)) using cDNA prepared from rat striatal mRNA and 1 μM each of thefollowing primers:5′-GTCGACCCT(GT)T(GT)(CG)GCC(AC)TCA(GT)CA(TC)(GA)G(AG)-TCGCTA-3′ SEQ IDNO: 1 5′-AAGCTTATGAA(AG)AAGGGCAG(GC)CA(AG)CAGAGG(CT)(CT)-(AG)(CA)A- 3′.SEQ ID NO: 2

[0057] The PCR timing cycle was 1.5 min at 93° C., 2 min at 50° C., and4 min at 72° C. followed by a 7 min extension at 72° C. The reactionproducts were purified and size fractionated on a 1% agarose gel.Individual bands were excised, electroeluted, concentrated byultrafiltration, and ligated into the pCR1000© vector (INVITROGEN).Competent INVaF bacterial cells (INVITROGEN) were transformed andmini-preparations of plasmid DNA prepared for insert sequencing.

[0058] This process resulted in the amplification of several cDNAfragments ranging from 300 to approximately 500 base pairs (bp) in size.Each fragment was preliminarily characterized by DNA sequence analysis.Several of these cDNA fragments exhibited sequence homology withpreviously cloned G-protein coupled receptors such as serotonin, one ofwhich, clone St-B17₁ (538 bp), was particularly homologous with severalcatecholamine receptors and was thus selected for furthercharacterization.

[0059] We selected 1×10⁶ recombinants from the rat striatal cDNAlibrary, constructed in the λ ZAP II© vector (STRATAGENE, La Jolla,Calif.), to be screened with the 538 bp St-B17₁ PCR fragment. The 538 bpfragment was made into a probe by ³²P-labeling (via nick translation)using well known methods. Duplicate nitrocellulose filters werehybridized in 50% formamide, 0.75 M NaCl, 0.075 M sodium citrate(5×SSC), 5×Denhardt's solution 0.02 M Na₂HPO₄, 0.25% SDS, 0.15 mg/mlsalmon sperm DNA, and 4×10⁶ dpm/ml of ³²P-labeled probe for 24 hr at 37°C. High stringency washing of the filters was performed with 1×SSC and0.1% SDS at 65° C. prior to autoradiography.

[0060] The λ phage found to hybridize with the probe were subsequentlyplaque purified. In vivo excision and rescue of the nested pBluescriptplasmids from the λ ZAP II clones was performed using helper phageaccording to the STRATAGENE protocol.

[0061] Briefly, the λ phage carry a nested pBluescript plasmid which canbe excised via the STRATAGENE protocol. This nested plasmid carried theSt-B17 gene and could be used for subsequent sequencing andtransfection. Nucleotide sequence analysis was performed using theSanger dideoxy nucleotide chain termination method with Sequenase© (USBIOCHEMICAL CORP.) on the excised denatured double-stranded plasmidtemplates.

[0062] One positive clone (St-B17₂) from the above screening, containinga cDNA insert of 2.8 kb, was isolated and nucleotide sequenced. Thelargest open reading frame was 879 bp long encoding a protein containingsequences homologous to transmembrane (TM) regions I-VI of previouslycloned biogenic amine receptors.

[0063] Further analysis of the 2.8 kb cDNA revealed the presence of asequence 210 bp beyond the end of the open reading frame which washomologous to the seventh transmembrane (TM) region of severalreceptors. Careful examination of the DNA sequence between theseputative TM regions VI and VII revealed the presence of consensus donorand acceptor sequences for intron/exon splice junctions suggesting thepossibility that this region represented an un-spliced intron having aninframe stop codon. Sequence ID NO: 9 is a partial polynucleotidesequence of the 2.8 kb clone having the entire coding sequence, and theinternal intron. The amino acid sequence corresponding to the firstcoding region of SEQUENCE ID NO:9 is shown as SEQUENCE ID NO:10. Theamino acid sequence corresponding to the second coding region ofSEQUENCE ID NO:9 is shown as SEQUENCE ID NO:11.

[0064] This would explain the homology to the seventh TM region in thepurported non-coding portion of the gene. The possibility that we hadisolated a cDNA from a partially spliced mRNA was examined, as describedin Example 2 below, using PCR to amplify this putative intron regionfrom cDNA prepared from rat striatal mRNA.

EXAMPLE 2

[0065] Amplification and Analysis of the 2.8 kb Insert

[0066] Primers P3 and P4 were synthesized to have homology near to the3′ and 5′ ends of the putative splice region. The sequence of primer P4is complementary to the coding sequence since it was designed to annealwith the homologous DNA strand. With these primers, we would eitherisolate 223 bp sequences corresponding to a spliced message, or 416 bpsequences corresponding to an unspliced message. This system couldthereby indicate if the putative intron acceptor/donor sites were reallyutilized for splicing reactions in the cell. SEQ ID PRIMER SEQUENCENUMBER P3 AAGCATAGCA GGAAGGCCTT GAAGGCCAGC CTG SEQ ID NO: 3 P4GGCGAGAAAT ACGCCCTGAA GTTCTCCCGG GAC SEQ ID NO: 4

[0067] Amplification across the putative splice region with primers P3and P4 resulted in a primary DNA product of 223 bp, the size predictedfor mRNA spliced at the observed donor/acceptor sites, and a minor bandof 416 bp, the size expected based on the sequence of the unsplicedSt-B17₂ cDNA clone. We thus believed that the St-B7₂ cDNA clone wasderived from a minor, incompletely spliced mRNA.

[0068] Since an additional screen of the cDNA library did not result inthe isolation of a full-length, completely spliced clone, we used PCRfollowing the protocol outlined in Example 1 to amplify the predictedcoding sequence from rat striatal mRNA.

EXAMPLE 3

[0069] Amplification of Putative Coding Region

[0070] We used primers P1 and P2 (shown below as SEQ ID NOS: 5 and 6,respectively) and PCR to amplify the 2.8 kb clone isolated in Example 2from the rat striatum cDNA library. Primer P2 was complementary to thecoding sequence since it was designed to hybridize with the homologousnucleotide strand. PRI- MER SEQUENCE SEQ ID NUMBER P1 TTGCCAATACTACTCTAAGG TGCAGCTTCC SEQ ID NO: 5 P2 CACACGACTT AACTCCATAG AGTCGATCGGSEQ ID NO: 6

[0071] Two nucleotide sequences of 1.6 and 1.4 kb were obtained from thePCR reaction, the latter of which (St-B17₃) was subcloned into plasmidpBluescript II© SK (+). Sequence analysis confirmed the identity ofSt-B17₃ and revealed that the putative intron was absent in this clone(SEQ ID NO: 7) with the flanking exons spliced together at nucleotide873 as predicted. Splicing at this position resulted in a 1,311 bp openreading frame encoding a protein of 437 amino acid residues (SEQ ID NO:8) with a calculated molecular weight of 46.8 kD.

[0072] Hydropathy analysis of the deduced amino-acid sequence of thespliced St-B17₃ protein indicated seven hydrophobic regions predicted torepresent putative transmembrane spanning domains. When compared topreviously cloned G-protein coupled receptors, the transmembrane regionsof St-B17 exhibited high homology to various serotonin (5-HT) receptorssuggesting that it may be a member of this receptor family.

[0073] Within the transmembrane regions, St-B17₃ exhibited homologies of41%, 40%, 39%, 38%, 37% and 36% with 5-HT₂, 5-HT_(1D), 5-HT_(1C),5-HT_(1B), 5-HT_(1A) and 5-HT_(1E) receptors, respectively. The thirdcytoplasmic loop of St-B17₃, consisting of 57 residues, would be theshortest of the cloned 5-HT receptors, though similar in length to the5-HT_(1C) and 5-HT₂ receptors. These receptors have been describedpreviously by Julius, D. et al., Science 241, 558-564 (1988); andJulius, D. et al., Proc. Natl. Acad. Sci. USA 87, 928-932 (1990); andPritchett, D. B., et al., EMBO J. 7, 4135-4140 (1988). Similarly, whilethe 117 residue carboxy-terminal tail of St-B17₃ is the longest amongstthe 5-HT receptors, it is similar in length to that found in the5-HT_(1C) and 5-HT₂ receptors.

[0074] The combination of a relatively short third cytoplasmic loop anda long carboxy-terminus is common among receptors which are coupled tothe stimulation of either the adenylyl cyclase or phospholipase C signaltransduction systems. St-B17₃ also contained one potential N-linkedglycosylation site at Asn-9 in the extracellular amino terminus.Additionally, several potential sites for phosphorylation by the cyclicAMP-dependent protein kinase or protein kinase C in both the thirdcytoplasmic loop and the intracellular carboxy-terminal tail werededuced.

[0075] To definitively establish the identity of the receptor encoded bySt-B17₃, we wanted to express this gene in mammalian cells.Unfortunately, the PCR-amplified full-length, correctly spliced St-B17₃cDNA could not be used directly for expression due to severalPCR-generated base substitutions in the 5′ region up-stream of theinternal Bam H1 site.

[0076] We thus constructed a hybrid cDNA in which the entire 3′ sequencedown-stream of the Bam H1 site from the intron-containing clone(St-B17₂) was replaced with the corresponding Bam H1 fragment from thecorrectly spliced, PCR-amplified cDNA (St-B17₃) as explained below inExample 4.

EXAMPLE 4

[0077] Construction of Expression Clone

[0078] We cleaved the 1.4 kb St-B17₃ clone isolated in Example 3 withBamHI by well known methods. We also cleaved the 2.8 kb St-B17₂ cloneisolated in Example 1 with BamHI. By mixing and annealing the fragmentsfrom these two digestions, we derived a clone (St-B17) with the 5′ endof St-B17₂ and the 3′ end of St-B17₃. The resultant fusion gene had afunctional 5′ leader and initiation codon, and a correctly splicedintron/exon junction.

[0079] This construct was subcloned into the eucaryotic expressionvector pCD-Srα yielding pSRα-B17. This clone was then used to transfectmammalian cells to study the expression, binding, and regulation of thenew serotonin receptor gene as discussed below in Example 5.

EXAMPLE 5

[0080] Transfection of Mammalian Cells with St-B17

[0081] Transient transfection of Cos-7 cells with pSRα-B17 resulted inthe appearance of high affinity and saturable binding sites for theserotonergic ligand [¹²⁵I]-LSD (lysergic acid diethylamide).

[0082] Cos-7 cells were transfected with the pSRα-B17 construct usingthe calcium phosphate precipitation method as previously described byMonsma, F. J. et al., Proc. Natl. Acad. Sci. USA 87, 6723-7 (1990).Cells were harvested 72 hours after transfection and either disrupted ina dounce homogenizer in 50 mM Tris-HCl, pH 7.4 at 37° C., 10 mM MgSO₄and 0.5 m M EDTA, or frozen in 5 mM Tris-HCl, pH 7.4 at 25° C., 5 mMMgCl₂, 250 mM sucrose and stored in liquid N₂ prior to membranepreparation. Crude membranes were prepared from cell homogenates bycentrifugation at 43,000×g, and re-suspension in homogenization bufferat a protein concentration of 60 μg/ml.

[0083] Once the crude membranes had been prepared from the cellhomogenates, we proceeded to pharmacologically screen various drugs fortheir effect on [¹²⁵I]-LSD to St-B17 as discussed below in Example 6.

EXAMPLE 6

[0084] Pharmacological Screening Assays Using St-B17

[0085] It was first necessary to determine the binding strength andsaturation point of LSD, so that we could later analyze drugs thatdisplaced this chemical. Radiolabeled LSD ([¹²⁵I]-LSD) exhibited adissociation constant (K_(D)) of 1.26 (±0.17, n=3) nM and maximumbinding (B_(max)) values ranging from 2-5 pMoles/mg protein (FIG. 1)when added to the membranes prepared by the method of Example 5. FIG. 1shows the saturation analysis of [¹²⁵I]-LSD binding to membranesprepared from Cos-7 cells transiently transfected with constructpSRα-B17. The experiment reported is representative of 3 independentexperiments conducted in triplicate. The inset (FIG. 1) is a scatchardanalysis of the saturation binding data. In this experiment the K_(D)and B_(max) values were 1.5 nM and 3.4 pMoles mg protein⁻¹,respectively. This assay shows the strong binding of LSD to the St-B17receptor.

[0086]FIG. 2 displays the results of a pharmacological competition assayto analyze the displacement of [¹²⁵I]-LSD in the presence of variousserotonin receptor binding drugs. This experiment was performed on Cos-7cell membranes expressing the St-B17 serotonin receptor. The competitioncurves shown are representative of 3 independent experiments conductedin triplicate. Average inhibition constants (K_(i)) and SEM values aregiven in Table 1. No specific binding of [¹²⁵I]-LSD was observed inuntransfected Cos-7 cells or in cells transfected with the pCD-Srαexpression vector alone (controls). TABLE 1 Pharmacology of St-B17Expressed in Cos-7 Cells K_(i) (nM) Mean ± SEM Drug vs. [¹²⁵I]-LSD vs.[³H]-5-HT Methiothepin  1.84 ± 0.23  0.39 ± 0.063 Lisuride  8.19 ± 0.485.3 ± 1.3 Clozapine 12.87 ± 1.69  20 ± 2.5 Dihydroergotamine 13.07 ±0.90 5.4 ± 5.4 2-Br-LSD 17.14 ± 1.11 Pergolide 29.87 ± 7.31 Metergoline29.97 ± 0.38 61 ± 23 Amoxipine 30.40 ± 1.64 Lergotrile 36.45 ± 1.045-methoxytryptamine 38.92 ± 3.95 18 ± 2  Ritanerin 44.12 ± 4.35 16 ± 2 Mianserin 45.74 ± 8.93 38 ± 7  Clomiprimine 53.78 ± 3.285-Hydroxy-N,ω-methyltryptamine 57.95 ± 8.32 Loxapine 64.75 ± 0.60  56 ±1.5 Amitriptyline 69.64 ± 7.21 82 ± 6  N,N-Dimethyl-5-methoxytryptamine79.81 ± 3.86 1(1-naphthyl)piperazine 103.61 ± 13.815-Benzyloxytryptamine 110.18 ± 17.90 Cyproheptadine 133.85 ± 6.17 Doxepin 135.74 ± 7.11  Nortriptyline 147.49 ± 2.61  5-HT 151.01 ± 12.7856 ± 9  Dihydroergocryptine 160.70 ± 13.21 Imipramine 208.49 ± 22.76 190± 3  N,ω-Methyltryptamine 341.60 ± 32.21 MethyscrGide 371.74 ± 72.66Tryptamine 438.27 ± 14.84 TFMPP 482.37 ± 37.21 CGS 12066B 737.69 ± 66.635-Carboxamidotryptamine 774.35 ± 84.39 253 ± 20  PAPP 805.62 ± 75.17Mesulergine 1718.88 ± 248.35 Fluoxetine 1765.36 ± 290.37 mCPP 2309.60 ±343.05 2-MPP 3054.95 ± 321.06

[0087] Increasing concentzations of the indicated compounds were used toinhibit the binding of either 0.5 nM [¹²⁵I]-LSD or 5 nM (³H)-5-HT tomembranes of Cos-7 cells transiently transfected with clone St-B17.[¹²⁵I]-LSD competition assays were performed by incubating thetransfected Cos-7 cells with [¹²⁵I]-LSD and the indicated concentrationsof each drug as described above and in FIG. 2, while those for [³H]-5-HTwere performed as previously described (Hamblin, M. W. & Metcalf, M. A.,Mol. Pharmacol. 40, 143-148 (1991)). K_(i) values were obtained fromgraphically determined IC₅₀ values by the method of Cheng and Prussoff(Biochem. Pharmacol. 22, 3099-3108 (1973)) and are presented as thegeometric mean±SEM (n=3) calculated according to De Lean et al. (Mol.Pharmacol. 21, 5-16 (1982)).

[0088] This method thereby provides a way of screening drug candidatesfor central nervous system activity by testing its binding to isolatedSt-B17. We believe that drugs which bind to the St-B17 receptor in vitrowill also have effects in vivo. In addition to directly measuring thebinding of a drug to the St-B17 receptor, we were also able to measurethe displacement of known binding ligands to the St-B17 receptor. Bymeasuring the binding of a ligand as discussed above, and then comparingthat binding to the level of binding in the presence of a drugcandidate, we were able to estimate the potential of the drug todisplace the ligand.

[0089] The following compounds were screened and found to exhibitinhibition constant (K_(i)) values>10 μM: zimelidine, zacopride,NAN-190, citalopram, BIMU 1, metoclopromide, fenfluramine, MDL 72222,8-OH-DPAT, BRL 24924, BRL 43694A, GR 38032F, ICS 205,930, ketanserin,melatonin, spiroperidol, tyramine, DAU 6215, DAU 6285, dopamine,epinephrine, histamine, idazoxan, LY 278,584, maxindol, norepinephrine,octopamine, paroxetine, and pindolol.

[0090] Preliminary characterization of the St-B17 pharmacology indicatedthat amongst several endogenous biogenic amines, including dopamine,melatonin, epinephrine, norepinephrine or histamine, only 5-HT(serotonin) was capable of completely displacing [¹²⁵I]-LSD binding,exhibiting a K_(i) of 150 nM (FIG. 2 and Table 1). The Hill coefficientfor the 5-HT competition curve was not significantly different fromunity and the affinity of 5-HT was not influenced by addition of theguanine nucleotide analogue GppNHp.

[0091] The binding of 5-HT to St-B17 was also investigated directlyusing [³H]-5-HT as the radioligand. Saturation analysis of [³H]-5-HTbinding revealed the presence of a single class of high affinity,saturable binding sites in transfected, but not untransfected, Cos-7cells. Analysis of the [³H]-5-HT saturation binding data revealed aK_(D) of 37 (±5.0, n=3) nM and B_(max) values of 1-3 pMoles/mg protein.The binding of [³H]-5-HT was similarly not affected by the addition ofguanine nucleotides. These initial binding data would thus suggest thatSt-B17 encodes a 5-HT receptor subtype.

[0092] Further characterization of the St-B17 pharmacology involved theutilization of a variety of drugs which are known to exhibit specificityfor various serotonergic receptor subtypes and other binding sites. Theaverage K_(i) values for compounds competing with better than 10 μMaffinity are shown in Table 1 with representative competition curves for[¹²⁵I]-LSD binding shown in FIG. 2.

[0093] Examination of the rank order of potency for a variety ofserotonergic agents reveals that the pharmacology of clone St-B17 doesnot correspond to any previously described serotonin receptor subtype. Anumber of drugs selective for 5-HT₃ and 5-HT₄ receptors (i.e., MDL72222, ICS 205,930, or DAU 6285) exhibit virtually no affinity forSt-B17, while agents selective for other 5-HT receptor subtypes such as8-OH-DPAT (5HT_(1A)), CGS 12066B (5-HT_(1B)), mesulergine (5-HT_(1C)),or ketanserin (5-HT₂) bind with relatively low affinity. Ergotalkaloids, especially ergoline derivatives (i.e., LSD, lisuride, orpergolide), display relatively high affinity for St-B17 as does thenon-selective serotonergic antagonist methiothepin.

[0094] Interestingly, the atypical and typical anti-psychotics clozapineand loxapine, respectively, also exhibited high affinity for St-B17, asdid several tricyclic anti-depressant drugs (i.e., amoxipine,clomiprimine, and amitriptyline) which all had K_(i) values under 100nM. In general, the drugs which exhibited the greatest affinity forSt-B17 (i.e., K_(i)<100 nM) were tricyclic, ergoline or tryptaminederivatives.

[0095] Competition for [³H]-5-HT binding by a number of drugs revealed,with a few exceptions, the same rank order of potency as for inhibitionof [¹²⁵I]-LSD binding (Table 1). However, for some drugs the K_(i)values determined by competition with [³H]-5-HT were up to 5-fold lowerthan those determined by competition with [¹²⁵I]-LSD, with the exceptionof clozapine, metergoline and amitriptyline, which exhibited somewhatgreater potency in competition with [¹²⁵I]-LSD.

[0096] Although the pharmacological profile of St-B17 did not correspondto previously defined 5-HT receptor subtypes, it did resemble theprofile described by Conner and Mansour for 5-HT stimulation of adenylylcyclase activity in the NCB-20 neuroblastoma cell line (Mol. Pharmacol.(1990) 37, 742-751). In these cells 5-HT activated adenylyl cyclase withan EC₅₀ of 300 nM and a variety of tryptamine derivatives exhibit EC₅₀values similar to their K_(i) values for inhibition of [¹²⁵I]-LSDbinding to St-B17. Similarly, antagonism of 5-HT-stimulated adenylylcyclase activity in NCB-20 cells by a variety of compounds, includingmethiothepin, mianserin, amitriptyline, and cyproheptadine, alsoexhibited apparent K_(i) values and a rank order of potency similar tothose observed for inhibition of [¹²⁵I]-LSD binding to St-B17.

[0097] It is contemplated that other means of labeling LSD or the othertested drugs would also provide an assay for the inhibition of compoundssuch as that discussed above. LSD could be labeled either radioactivelyor colormetrically and still provide the desired assay.

[0098] The distribution of mRNA encoding St-B17 was evaluated byNorthern blot analysis of poly (A+) RNA prepared from a variety of ratbrain regions as well as other peripheral tissues. The results fromthese experiments are shown below in Example 7.

EXAMPLE 7

[0099] Distribution of mRNA Encoding St-B17

[0100]FIG. 3 shows a Northern blot analysis of St-B17 RNA transcripts inrat brain. Each lane contained 3 μg of poly (A+) RNA. Lanes: AMYG,amygdala; CB, cerebellum; CTX, cerebral cortex; HIP, hippocampus; HYP,hypothalamus; MED, medulla; OB, olfactory bulb; OT, olfactory tubercle;PIT, pituitary; RET, retina; STR, striatum; THAL, thalamus. Locations ofRNA size (kb) markers are indicated.

[0101] To prepare the Northern blot, poly (A+) RNA was prepared from ratbrain regions using the FASTTRACK© mRNA isolation system (INVITROGEN). 3μg of poly (A+) RNA was denatured and subjected to electrophoresis on a1% agarose gel containing 0.66 M formaldehyde. RNA was transferred to anylon membrane (Genescreen Plus, Dupont) by capillary transfer andimmobilized by UV crosslinking. Northern blots were probed with theentire 2.8 kb cDNA insert of clone St-B17 [³²P]-labeled by the randomprimer method. Northern blots were hybridized with 3×10⁶ dpm/ml of probein 1 M NaCl, 1% SDS, 10% dextran sulfate at 60° C. for 18 hr. Blots werewashed in 2×SSC/1% SDS at room temperature, followed by 0.2×SSC/1% SDSat 65° C., and were exposed with an intensifying screen for 15 days at−70° C.

[0102] A single transcript of 4.2 kb was observed in various brainregions with highest expression appearing to occur in the corpusstriatum (FIG. 3). St-B17 mRNA was also observed in the amygdala,cerebral cortex and olfactory tubercle whereas it was absent, or presentin undetectable levels, in the cerebellum, hippocampus, hypothalamus,medulla, olfactory bulb, pituitary, retina and thalamus. Similarly, notranscript was observed, even after prolonged exposures, in Northernblots of mRNA from rat heart, lung, kidney, spleen, pancreas, smoothmuscle, skeletal muscle, stomach, ovary, prostrate, or testes (data notshown).

[0103] This result indicates that the St-B17 receptor is produced inmuch higher levels in the corpus striatum than in other regions of thebrain. In addition, the receptor is undetectable in non-brain tissues.

[0104] The localization of St-B17 mRNA to limbic and cortical regions,and the relatively potent interaction of several therapeuticallyimportant drugs, including the atypical anti-psychotic clozapine, thetypical anti-psychotic loxapine, and the tricyclic anti-depressantsamoxipine, clomiprimine, amitriptyline, and nortriptyline with St-B17suggests that this receptor may play an important though hithertounappreciated role in several neuropsychiatric disorders which involveserotonergic systems.

[0105] In addition to the mRNA distribution of St-B17, we were alsointerested in the intracellular cascade of events following agonistbinding to the receptor. One possible mechanism of receptor signalinginvolved adenylate cyclase activity. To investigate this possibility,the following experiment was performed.

EXAMPLE 8

[0106] Analysis of Receptor Mediated cAMP Activity

[0107] Human Embryonic Kidney cells (HEK 293 available from the ATCC)were stably co-transfected in 150mm dishes by the CaPO₄ technique with30 μg of pSRα-B17 and 3 μg of pMam-neo (INVITROGEN). These HEK 293 cellswere then subjected to selection with 600 μg/ml genetecin (G418)(GIBCO). The resistant colonies were collected and screened for receptorexpression by [¹²⁵I]-LSD binding as described in Example 6. One clonewas isolated which expressed approximately 800 fMoles/mg St-B17 receptorprotein and was subsequently used for assessment of cAMP accumulation.

[0108] Intracellular cAMP levels were determined after a 5 minuteincubation of intact cells with various test compounds at 37° C. in thepresence of the cAMP phosphodiesterase inhibitor RO-20-1724, using amodification of a previously described cAMP assay (Monsma, F. J., Jr.,Barton, A. C., and Sibley, D. R. (1990) J. Neurochemistry 54, 1200-1207)wherein the adrenal cAMP binding protein was replaced with 0.8 mg/mlprotein kinase A in water.

[0109] As shown in FIG. 4, serotonin causes a potent dose-dependentincrease in cAMP levels in transfected HEK-293 cells with an average 50%effective concentration (EC₅₀) of 145 (±40, n=3) nM, whereas there wasno detectable response in un-transfected cells (data not shown). Thissuggests that the St-B17 receptor mediates some part of its cellsignaling through a cAMP-related pathway.

[0110] A pharmacological analysis of the cAMP response (FIG. 5)indicates that the serotonergic agonists 5-MT and 5-CT are also able toelicit an increase in cAMP levels. The ergot alkaloids lisuride anddihydroergotamine also stimulate cAMP accumulation, although these drugsappear to function as partial agonists to the St-B17 receptor.Amoxipine, methiothepin and clozapine (FIG. 5) all appear to act asantagonists of this receptor as they had no significant effect on cAMPlevels on their own, but were able to substantially inhibit the responseelicited by 5-HT. These data suggest that the St-B17 receptor isfunctionally linked to the adenylyl cyclase signal transduction system.

[0111] The measurement of cAMP levels in a cell transfected with St-B17can thereby provide an assay for detecting potential agonists andantagonists of serotonin receptors. Using the above method, we cananalyze the accumulated level of cAMP in the cell after treatment with adrug to be tested. A lowering of the cAMP level in the presence of 5-HTindicates a drug which has inhibitory affects on 5-HT receptor binding.

[0112] We were also interested in isolating the human variation of thepresent invention. Our method of isolating the human receptor isdiscussed below.

EXAMPLE 9

[0113] Isolation of the Human St-B17 Receptor

[0114] Two different restriction fragments derived from the rat St-B17gene were used as probes to retrieve the human homolog. A 1192 base-pairXma I-BstXI fragment and a 655 base-pair Bam HI-EagI fragment of ratSt-B17 were labeled with [α³²P]-dCTP using the well knownnick-translation method. These labeled fragments were then used toscreen a commercially available human genomic library (Stratagene#946205).

[0115] The library was plated onto host cells C600 and PLK17respectively, and duplicate nylon (Nytran, Schleicher and Schuell) liftsmade. Each of the labeled fragments was hybridized (6×SSC at 55° C.,pH7) and washed (2×SSC, 58° C.) at moderate stringency. Positiveisolates were plaque purified through a second round of hybridization.

[0116] An insert from each positive plaque isolate was excised by EcoRIdigestion, and then subcloned into a corresponding site in the phagemidvector pBluescript SK(−) (Stratagene). These inserts were then digestedwith several restriction enzymes, and run on a Southern Blot. Fragmentshybridizing to labeled St-B17 rat sequences were identified by Southernhybridization. Hybridizing fragments were then subcloned intopBluescript SK(−) as discussed above. The nucleic acid sequence of theseinserts was then determined by dideoxynucleotide termination usingSequenase (United States Biochemicals) in conjunction with standardsubcloning techniques. The nucleotide sequence of the human St-B17 geneis SEQUENCE ID NO:12. The corresponding amino acid sequence is SEQUENCEID NO: 13.

[0117] It should be understood that all of the previous experimentsrelating to identification of binding ligands to the rat receptor can beperformed in a similar fashion with the human gene. For instance, thehuman gene can be cloned into an expression plasmid and placed in COS-7cells by methods similar to those discussed above, and used to assaybinding of competitors such as those revealed in Table 1. This wouldprovide a method of assaying inhibitors of serotonin binding to theSt-B17 receptor.

[0118] Specifically, Example 8 can be repeated with the human receptorto reveal if a particular compound was mimicking or inhibiting thebinding of serotonin by measurement of the relative level of cAMP beforeand after treatment with the compound. The methods of this example canalso be repeated with cDNA of other mammals or other vertebrates toobtain the corresponding St-B17 receptor from these organisms.

[0119] Other experiments are also contemplated relating to discoveringwhich cells in vivo express the receptor gene on their surface. To allowus the ability to study the expression of the receptor on cell surfacesthroughout the body, we needed to produce antibodies against thereceptor protein. A method for producing antibodies is discussed belowin Example 10.

EXAMPLE 10

[0120] Production of Antibodies Against St-B17

[0121] COS-7 cells expressing the St-B17 receptor protein derived inExamples 11 and 12 are lysed with NP40, and the isolated membranes areinjected into rabbits. The lysed membranes are isolated in a non-ionicdetergent so as not to affect the membrane bound receptors. Freundsadjuvant is used in the injection to help stimulate an antigenicresponse by the rabbits. After two booster shots of the lysed membranes,the rabbits are bled and the sera isolated by centrifugation.

[0122] The antibodies in the crude rabbit sera extract are ¹²⁵I labeledby well known methods, and tested for activity against the transfectedCOS-7 cells. A western blot having one lane containing proteins fromtransfected cell lysates, and a second lane having untransfected lysates(control) is run. A strong band indicating antibody binding at 46.8 kdin the transfected cell lane, that is not apparent in the untransfectedlane shows that polyclonal antibodies against the St-B17 receptorprotein have been properly isolated.

[0123] Monoclonal antibodies can be made by well known methods inaddition to the polyclonal antibodies discussed above. One method ofproducing monoclonal antibodies is discussed below in Example 11.

EXAMPLE 11

[0124] Production of Monoclonal Antibodies Against St-B17

[0125] The St-B17 transfected COS-7 cells produced in the previousexamples are lysed with NP-40 and the cell membranes are pelleted bycentrifugation. The isolated membranes, having bound St-B17 receptorproteins are injected in Freunds adjuvant into mice. After beinginjected 9 times over a three week period, the mice spleens are removedand resuspended in PBS.

[0126] The suspended spleen cells are mixed (approximately 4:1 with SP2/0 Myeloma cells. Polyethylene glycol is added to fuse the myelomacells to the spleen cells, and the fused cells are selected in HATmedia. The fused cells are aliquoted so that only one cell is grown ineach well of a 96 well microtiter plate. Each cell is grown and themedia removed and secreted proteins are ¹²⁵I labeled. The labeled mediafrom each well is used to probe a Western blot of transfected anduntransfected COS-7 cells (see Example 12). The desired fusion cell willproduce a monoclonal antibody that strongly binds a 46.8 kD band in thetransfected COS-7 cell lane on the Western blot, but doesn't bind to anyother protein in that lane, or the control lane.

[0127] This method provides a way of detecting expression of the St-B17serotonin receptor protein. Another method of detecting expression ofSt-B17 is by in situ hybridization as discussed below in Example 12.

EXAMPLE 12

[0128] In Situ Hybridization of St-B17

[0129] In situ hybridization allows the identification of mRNA withinintact tissues, such as the rat brain. In this method, oligonucleotidescorresponding to unique portions of the St-B17 gene (SEQ ID NO: 7) areused to detect specific mRNA species in the brain.

[0130] An anesthetized rat is transcardially perfused with cold PBS(5-20 minutes), followed by perfusion with a 4% formaldehyde solution.The brain is removed, frozen in liquid nitrogen, and cut into 5 μm to 30μm sections. The sections are placed on slides and incubated inproteinase K for approximately 15 minutes. The slides are then rinsed inDEP, water, and ethanol, and placed in a prehybridization buffer.

[0131] A radioactive probe corresponding to primer P1 is made by nicktranslation and incubated with the sectioned brain tissue. Afterincubation and air drying, the labeled areas are visualized byautoradiography. Dark spots on the tissue sample indicate hybridizationof the probe with the brain mRNA thereby demonstrating expression of theSt-B17 receptor.

[0132] All of the above cited references are herein incorporated byreference. The present invention should not be limited by these examplesbut only by the following claims.       v340      v350      v360      v370      v380      v390      v400       v410ACTCACCTCCCCCGGGGGGCGTGGTCAGTCGCGGTCTGTTCTCACGGACGGTCCCCGTCCAGCCTGCGCTTCGCCGGGGCACTCACCTCCCCCCGGGGGCGTGGTGAGTCGCGGTCTGTTCTCACG ACGGTCCCCGTCCAGCCTGCGTTCGCCGGGGCACTCACCTCCCCCGGGGGGCGTGGTGAGTCGCGGTCTGTTCTCACG-ACGGTCCCCGTCCAGCCTGCGCTTCGCCGGGGC         {circumflex over ( )}10       {circumflex over( )}20       {circumflex over ( )}30       {circumflex over( )}40        {circumflex over ( )}50       {circumflex over( )}60       {circumflex over ( )}70       v420      v430      v440       v450     v460      v470      v480       v490CCTCATCTGCTTTCCCGCCACCCTATCACTCCCTTGCCGTCCACCCTCGGTCCTCATGGTCCCAGAGCCGGGCCCAACCGCCTCATCTGCTTTCCCCCCACCCTATCACTCCCTTGCCGTCCACCCTCGGTCCTCATGGTCCCAGAGCCGGGCCCAACCGCCTCATCTGCTTTCCCGCCACCCTATCACTCCCTTGCCGTCCACCCTCGGTCCTCATGGTCCCAGAGCCGGGCCCAACCG{circumflex over ( )}80       {circumflex over ( )}90       {circumflexover ( )}100      {circumflex over ( )}110      {circumflex over( )}120      {circumflex over ( )}130      {circumflex over( )}140      {circumflex over ( )}150       v500      v510      v520       v530     v540      v550      v560        v570CCAATAGCACCCCGGCCTGGGGGGCAGGGCCGCC-GTCGGCCCCGGGGGGCAGCGGCTGGGTGGCGGCCGCG-CTGTGCGCCAATAGCACCCCGGCCTGGGGGGCAGG C GCC GTCG     GGGGGGCAGCGGCTGGCTGGCGGCCG GCTGTGCGCCAATAGCACCCCGGCCTGGGGGGCAGG-C-GCCCGTCGNNN--GGGGGGCAGCGGCTGGGTGGCGGCCG-GCCTGTGCG{circumflex over ( )}160      {circumflex over ( )}170      {circumflexover ( )}180        {circumflex over ( )}190        {circumflex over( )}200      {circumflex over ( )}210      {circumflex over( )}220       {circumflex over ( )}230         v580      v590      v600      v610      v620      v630      v640      v650TGGTCATCGCGCTGACGGCGGCGGCCAACTCGCTGCTGATCGCGCTCATCTGCACTCAGCCCGCGCTGCGCAACACGTCCTGGTCATCGCGCTGACGGCGGCGGCCAACTCGCTGCTGATCGCGCTCATCTGCACTCAGCCCGCGCTGCGCAACACGTCCTGGTCATCGCGCTGACGGCGGCGGCCAACTCGCTGCTGATCGCGCTCATCTGCACTCAGCCCGCGCTGCGCAACACGTCC     {circumflex over ( )}240      {circumflex over( )}250      {circumflex over ( )}260      {circumflex over( )}270      {circumflex over ( )}280      {circumflex over( )}290      {circumflex over ( )}300      {circumflex over ( )}310         v660      v670      v680      v690      v700      v710      v720      v730AACTTCTTCCTGGTGTCGCTCTTCACGTCTGACCTGATGGTGGGGCTGGTGGTGATGCCGCCGGCCATGCTGAACGCGCTAACTTCTTCCTGGTGTCGCTCTTCACGTCTGACCTGATGGTGGGCTGGTGGTGATGCCGCCGGCCATGCTGAACGCGCTAACTTCTTCCTGGTGTCGCTCTTCACGTCTGACCTGATGGTCOGGCTGGTGGTGATGCCGCCGGCCATGCTGAACGCGCT     {circumflex over ( )}320      {circumflex over( )}330      {circumflex over ( )}340      {circumflex over( )}350      {circumflex over ( )}360      {circumflex over( )}370      {circumflex over ( )}380      {circumflex over ( )}390         v740      v750      v760      v770      v780      v790      v800      v810GTACGGGCGCTGGGTGCTGGCGCGCGGCCTCTGCCTGCTCTGGACCGCCTTCGACGTGATGTGCTGCAGCGCCTCCATCCGTACGGGCGCTGGGTGCTGGCGCGCGGCCTCTGCCTGCTCTGGACCGCCTTCGACGTGATGTGCTGCAGCGCCTCCATCCGTACGGGCGCTGGGTGCTCGCGCGCGGCCTCTGCCTGCTCTGGACCGCCTTCGACGTGATGTGCTGCAGCGCCTCCATCC     {circumflex over ( )}400      {circumflex over( )}410      {circumflex over ( )}420      {circumflex over( )}430      {circumflex over ( )}440      {circumflex over( )}450      {circumflex over ( )}460      {circumflex over ( )}470         v820      v830      v840      v850      v860      v870      v880      v890TCAACCTCTGCCTCATCAGCCTGGACCGCTACCTGCTCATCCTCTCGCCGCTGCGCTACAAGCTGCGCATGACGCCCCTGTCAACCTCTCCCTCATCAGCCTGGACCGCTACCTGCTCATCCTCTCGCCGCTGCGCTACAAGCTGCGCATGACGCCCCTGTCAACCTCTGCCTCATCAGCCTGGACCGCTACCTGCTCATCCTCTCGCCGCTGCGCTACAAGCTGCGCATGACGCCCCTG     480       {circumflex over ( )}490      {circumflex over( )}500      510       {circumflex over ( )}520      {circumflex over( )}530      {circumflex over ( )}540      {circumflex over ( )}550         v900      v910      v920      v930      v940      v950      v960      v970CGTGCCCTGGCCCTAGTCCTGGGCGCCTGGAGCCTCGCCGCTCTCGCCTCCTTCCTGCCCCTGCTGCTGGGCTGGCACGACGTGCCCTGGCCCTAGTCCTGGGCGCTGGAGCCTCGCCGCTCTCGCCTCCTTCCTGCCCCTGCTGCTGGGCTGGCACGACGTGCCCTGGCCCTAGTCCTGGGCGGCTGGAGCCTCGCCGCTCTCGCCTCCTTCCTGCCCCTGCTGCTGGGCTGGCACGA     {circumflex over ( )}560      {circumflex over( )}570      {circumflex over ( )}580      {circumflex over( )}590      {circumflex over ( )}600      {circumflex over( )}610      {circumflex over ( )}620      {circumflex over ( )}630         v980      v990      v1000     v1010     v1020     v1030     v1040     v1050GCTGGGCCACGCACGGCCACCCGTCCCTGGCCAGTGCCGCCTGCTGGCCAGCCTGCCTTTTGTCCTTGTGGCGTCGGGCCGCTGGGCCACGCACGGCCACCCGTCCCTGGCCAGTGCCGCCTGCTGGCCAGCCTGCCTTTTGTCCTTGTGGCGTCGGGCCGCTGGGCCACGCACGGCCACCCGTCCCTGGCCAGTGCCGCCTGCTGGCCAGCCTGCCTTTTGTCCTTGTGGCGTCGGGCC     {circumflex over ( )}640      {circumflex over( )}650      {circumflex over ( )}660      {circumflex over( )}670      {circumflex over ( )}680     {circumflex over( )}690       {circumflex over ( )}700      {circumflex over ( )}710         v1060     v1070     v1080     v1090     v1100     v1110     v1120     v1130TCACCTTCTTCCTGCCCTCGGGTGCCATATGCTTCACCTACTGCAGGATCCTGCTAGCTGCCCGCAAGCAGGCCGTGCAGTCACCTTCTTCCTGCCCTCGGGTGCCATATGCTTCACCTACTGCAGGATCCTGCTAGCTGCCCGCAAGCAGGCCGTGCAGTCACCTTCTTCCTGCCCTCGGGTGCCATATGCTTCACCTACTGCAGGATCCTGCTAGCTGCCCGCAAGCAGGCCGTGCAG     {circumflex over ( )}720      {circumflex over( )}730      {circumflex over ( )}740      {circumflex over( )}750      {circumflex over ( )}760      {circumflex over( )}770      {circumflex over ( )}780      {circumflex over ( )}790         v1140     v1150     v1160     v1170     v1180     v1190     v1200GTGGCCTCCCTCACCACCGGCATGGCCAGTCAGGCCTCGGAGACGCTGCAGGTGCCCAGGACCCCACGCC-CAGGGGTGGGTGGCCTCCCTCACCACCGGCATGGCCAGTCAGGCCTCGGAGACGCTGCAGGT CCCAGGA CCCAGC  CAGGGGTGGGTGGCCTCCCTCACCACCGGCATGGCCAGTCAGGCCTCGGAGACGCTGCAGGTACCCAGGAGCCCA-GCGGCAGGGGTGG     {circumflex over ( )}800      {circumflex over( )}810      {circumflex over ( )}820      {circumflex over( )}830      {circumflex over ( )}840      {circumflex over( )}850      {circumflex over ( )}860      {circumflex over ( )}870v1210     v1220     v1230     v1240      v1250     v1260     v1270     v1280AGTCTGCTGACAGCAGGCGTCTAGCCACGAAGCA-CAGCAGGAAGGCCCTGAAGGCCAGCCTGACGCTGGGCATCCTGCTAGTCTGCTGACAGCAGGCGTCTAGC ACGAAG A CAGCAGGAAGG CCTGAAGGCCAGCTGACGCTGGGCATCCTGCTAGTCTGCTGACAGCAGGCGTCTAGCAACGAAG-AGCAGCAGGAAGGGCCTGAAGGCCAGCATGACGCTGGQCATCCTGCT      {circumflex over ( )}880      {circumflex over( )}890      {circumflex over ( )}900       {circumflex over( )}910      {circumflex over ( )}920      {circumflex over( )}930      {circumflex over ( )}940      {circumflex over ( )}950 v1290     v1300     v1310     v1320     v1330     v1340     v1350     v1360GGGCATGTTCTTTGTGACCTGGTTGCCCTTCTTTGTGGCCAACATAGTCCAGGCCGTGTGCGACTGCATCTCCCCAGGCCGGGCATGTTCTTTGTGACCTGGTTGCCCTTCTTTGTGGCCAACATAGTCCAGGCCGTGTGCGACTGCATCTCCCCAGGCCGGGCATGTTCTTTGTGACCTGGTTGCCCTTCTTTGTGGCCAACATAGTCCAGGCCGTGTGCGACTGCATCTCCCCAGGCC       {circumflex over ( )}960      {circumflex over( )}970      {circumflex over ( )}980      {circumflex over( )}990      1000      {circumflex over ( )}1010     {circumflex over( )}1020     {circumflex over ( )}1030 v1370     v1380     v1390     v1400     v1410     v1420     v1430     v1440TCTTCGATGTCCTCACATGGCTGGGTTACTGTAACAGCACCATGAACCCCATCATCTACCCACTCTTCATGCGGGACTTCTCTTCGATGTCCTCACATGGCTGGGTTACTGTAACAGCACCATGAACCCCATCATCTACCCACTCTTCATGCGGACTTCTCTTCGATGTCCTCACATGGCTGGGTTACTGTAACAGCACCATGAACCCCATCATCTACCCACTCTTCATGCFGGACTTC       {circumflex over ( )}1040     {circumflex over( )}1050     {circumflex over ( )}1060     {circumflex over( )}1070     {circumflex over ( )}1080     {circumflex over( )}1090     {circumflex over ( )}1100     {circumflex over ( )}1110 v1450     v1460     v1470     v1480     v1490     v1500     v1510     v1520AAGCGGGCGCTGGGCAGGTTCCTGCCATGTCCACGCTGTCCCCGGGAGCGCCAGGCCAGCCTGGCCTCGCCATCACTGCGAAGCGGGCGCTGGGCAGGTTCCTGCCATGTCCACGCTGTCCCCGGGAGCCCAGGCCAGCCTGGCCTCGCCATCACTGCGAAGCGGGCGCTGGGCAGGTTCCTGCCATGTCCACGCTGTCCCCGGGAGC-CCAGGCCAGCCTGGCCTCGCCATCACTGCG       {circumflex over ( )}1120     {circumflex over( )}1130     {circumflex over ( )}1140     {circumflex over( )}1150     {circumflex over ( )}1160      {circumflex over( )}1170     {circumflex over ( )}1180     {circumflex over ( )}1190 v1530     v1540     v1550     v1560     v1570     v1580     v1590     v1600CACCTCTCACAGCGGCCCCCGGCCCGGCCTTAGCCTACAGCAGGTGCTGCCGCTGCCCCTGCCGCCGGACTCAGATTCGGCACCTCTCACAGCGGCCCCCGGCCCGGCCTTAGCCTACAGCAGGTGCTGCCGCTGCCCCTGCCGCCGGACTCAGATTCGGCACCTCTCACAGCGGCCCCCGGCCCGGCCTTAGCCTACAGCAGGTGCTGCCGCTGCCCCTGCCGCCGGACTCAGATTCGG        {circumflex over ( )}1200     {circumflex over( )}1210     {circumflex over ( )}1220     {circumflex over( )}1230     {circumflex over ( )}1240     {circumflex over( )}1250     {circumflex over ( )}1260     {circumflex over ( )}1270 v1610     v1620     v1630     v1640     v1650     v1660     v1670     v1680ACTCAGACGCAGGCTCAGGCGGCTCCTCGGGCCTGCGGCTCACGGCCCAGCTGCTGCTTCCTGGCGAGGCCACCCAGGACACTCAGACGCAGGCTCAGGCGGCTCCTCGGGCTGCCGCTCACGGCCCAGCTGCTGCTTCCTGGCGAGGCCACCCAGGACACTCAGACGCAGGCTCAGGCGGCTCCTCCGGCGTGCCGCTCACGGCCCAGCTGCTGCTTCCTGGCGAGGCCACCCAGGAC        {circumflex over ( )}1280     {circumflex over( )}1290     {circumflex over ( )}1300     {circumflex over( )}1310     {circumflex over ( )}1320     {circumflex over( )}1330     {circumflex over ( )}1340     {circumflex over ( )}1350 v1690     v1700     v1710     v1720     v1730      v1740     v1750     v1760CCCCCGCTGCCCACCAGGGCCGCTGCCGCCGTCAATTTCTTCAACATCG-ACCCCGCGGAGCCCGAGCTGCGGCCGCATCCCCCCGCTGCCCACCAGGGCCGCTGCCGCCGTCAATTTCTTCAACATCGACCCCGCGGAGCCCGAGCTGCGGCCGCATCCCCCCGCTGCCCACCAGGGCCGCTGCCGCCGTCAATTTCTTCAACATCGSACCCCGCGGAGCCCGAGCTGCGGCCGCATC        {circumflex over ( )}1360     {circumflex over( )}1370     {circumflex over ( )}1380     {circumflex over( )}1390     {circumflex over ( )}1400     {circumflex over( )}1410     {circumflex over ( )}1420     {circumflex over ( )}1430 v1770      v1780     v1790     v1800     v1810     v1820     v1830     v1840CACTTGGCATCCCCACGAACTGACCCGGGCTTGGGGCTGGCCAATGGGGAGCTGGATTGAGCAGAACCCAGACCCTGAGTCACTTGGCATCCCCACGAACTGACCCGGCTTGGGGCTGGCCAATGCGGAGCTGGATTGAGCAGAACCCAGACCCTGAGTCACTTGGCATCCCCACGAACTGACCC-GGCTTGGGGCTGGCCAATGGGGACCTGGATTGAGCAGAACCCAGACCCTGAGT        {circumflex over ( )}1440     {circumflex over( )}1450      {circumflex over ( )}1460     {circumflex over( )}1470     {circumflex over ( )}1480     {circumflex over( )}1490     {circumflex over ( )}1500     {circumflex over ( )}1510  v1850     v1860     v1870     v1880     v1890     v1900     v1910     v1920CCTTGGGCCAGCTCTTGGCTAAGACCAGGAGGCTGCAAGTCTCCTAGAAGCCCTCTGAGCTCCAGAGGGGTGCG-CAGAGCCTTGGGCCAGCTCTTGGCTAAGACCAGGAGGCTGCAAGTCTCCTAGAAGCCCTCTGAGCTCCAGAGGGGTGCGCAGAGCCTTGGGCCAGCTCTTGGCTAAGACCAGGAGGCTGCAAGTCTCCTAGAAGCCCTCTGAGCTCCAGAGGGGTGCGGCAGAG         {circumflex over ( )}1520     {circumflex over( )}1530     {circumflex over ( )}1540     1550      {circumflex over( )}1560     {circumflex over ( )}1570     {circumflex over( )}1580     {circumflex over ( )}1590   v1930     v1940     v1950     v1960     v1970     v1980CTGACCCCCTGCTGCCATCTCCAGGCCCCTTACCTGCAGGGATCATAGCTGACTCAGACTGACCCCCTGCTGCCATCTCCAGGCCCCTTACCTGCAGGGATCATAGCTGACT AGACTGACCCCCTGCTGCCATCTCCAGGCCCCTTACCTGCAGGGATCATAGCTGACT-AGA         {circumflex over ( )}1600     {circumflex over( )}1610     {circumflex over ( )}1620     {circumflex over( )}1630     {circumflex over ( )}1640      {circumflex over( )}800      {circumflex over ( )}810      {circumflex over( )}820     {circumflex over ( )}830       {circumflex over( )}840      {circumflex over ( )}850      {circumflex over( )}860      {circumflex over ( )}870v1210     v1220     v1230     v1240      v1250     v1260     v1270     v1280AGTCTGCTGACAGCAGGCGTCTAGCCACGAAGCA-CAGCAGGAAGGCCCTGAAGGCCAGCCTGACGCTGGGCATCCTGCTAGTCTGCTGACAGCAGGCGTCTAGC ACGAAG A CAGCAGGAAGG CCTGAAGGCCAGCTGACGCTGGGCATCCTGCTAGTCTGCTGACAGCAGGCGTCTAGCAACGAAG-AGCAGCAGGAAGGGCCTGAAGGCCAGCATGACGCTGGGCATCCTGCT      {circumflex over ( )}880      {circumflex over( )}890      {circumflex over ( )}900       {circumflex over( )}910      {circumflex over ( )}920      {circumflex over( )}930      {circumflex over ( )}940      {circumflex over ( )}950 v1290     v1300     v1310     v1320     v1330     v1340    v1350      v1360GGGCATGTTCTTTGTGACCTGGTTGCCCTTCTTTGTGGCCAACATAGTCCAGGCCGTGTGCGACTGCATCTCCCCAGGCCGGGCATGTTCTTTGTGACCTGGTTGCCCTTCTTTGTGGCCAACATAGTCCAGGCCGTGTGCGACTGCATCTCCCCAGGCCGGGCATGTTCTTTGTGACCTGGTTGCCCTTCTTTGTGGCCAACATAGTCCAGGCCGTGTGCGACTGCATCTCCCCAGGCC       {circumflex over ( )}960      {circumflex over( )}970      {circumflex over ( )}980      {circumflex over( )}990      {circumflex over ( )}1000     {circumflex over( )}1010     {circumflex over ( )}1020     {circumflex over ( )}1030 v1370     v1380     v1390     v1400     v1410     v1420     v1430     v1440TCTTCGATGTCCTCACATGGCTGGGTTACTGTAACAGCACCATGAACCCCATCATCTACCCACTCTTCATGCGGGACTTCTCTTCGATGTCCTCACATGGCTGGGTTACTGTAACAGCACCATGAACCCCATCATCTACCCACTCTTCATGCGGACTTCTCTTCGATGTCCTCACATGGCTGGGTTACTGTAACAGCACCATGAACCCCATCATCTACCCACTCTTCATGCTGGACTTC       {circumflex over ( )}1040     {circumflex over( )}1050     {circumflex over ( )}1060     {circumflex over( )}1070     {circumflex over ( )}1080     {circumflex over( )}1090     {circumflex over ( )}1100     {circumflex over ( )}1110 v1450     v1460     v1470     v1480     v1490     v1500     v1510     v1520AAGCGGGCGCTGGGCAGGTTCCTGCCATGTCCACGCTGTCCCCGGGAGCGCCAGGCCAGCCTGGCCTCGCCATCACTGCGAAGCGGGCGCTGGGCAGGTTCCTGCCATGTCCACGCTGTCCCCGGGAGCCCAGGCCAGCCTGGCCTCGCCATCACTGCGAAGCGGGCGCTGGGCAGGTTCCTGCCATGTCCACGCTGTCCCCGGGAGC-CCAGGCCAGCCTGGCCTCGCCATCACTGCG       {circumflex over ( )}1120     {circumflex over( )}1130     {circumflex over ( )}1140     {circumflex over( )}1150     {circumflex over ( )}1160      {circumflex over( )}1170     {circumflex over ( )}1180     {circumflex over ( )}1190 v1530     v1540     v1550     v1560     v1570     v1580     v1590     v1600CACCTCTCACAGCGGCCCCCGGCCCGGCCTTAGCCTACAGCAGGTGCTGCCGCTGCCCCTGCCGCCGGACTCAGATTCGGCACCTCTCACAGCGGCCCCCGGCCCGGCCTTAGCCTACAGCAGGTGCTGCCGCTGCCCCTGCCGCCGGACTCAGATTCGGCACCTCTCACAGCGGCCCCCGGCCCGGCCTTAGCCTACAGCAGGTGCTGCCGCTGCCCCTGCCGCCGGACTCAGATTCGG        {circumflex over ( )}1200     {circumflex over( )}1210     {circumflex over ( )}1220     {circumflex over( )}1230     {circumflex over ( )}1240     {circumflex over( )}1250     {circumflex over ( )}1260     {circumflex over ( )}1270 v1610     v1620     v1630     v1640     v1650     v1660     v1670     v1680ACTCAGACGCAGGCTCAGGCGGCTCCTCGGGCCTGCGGCTCACGGCCCAGCTGCTGCTTCCTGGCGAGGCCACCCAGGACACTCAGACGCAGGCTCAGGCGGCTCCTCGGGCTGCGGCTCACGGCCCAGCTGCTGCTTCCTGGCGAGGCCACCCAGGACACTCAGACGCAGGCTCAGGCGGCTCCTCGGGCGTGCGGCTCACGGCCCAGCTGCTGCTTCCTGGCGAGGCCACCCAGGAC         v10       v20       v30      v40    v50           v60       v70       v80       v90       v100      v110      v120MVPEPGPTANSTPAWGAGPPSAPGGSGWVAAALCVVIALTAAANSLLIALICTQPALRNTSNFFLVSLFTSDLMVGLVVMPPAMLNALYGRWVLARGLCLLWTAFDVMCCSASILNLCLI||||||||||||||||||:....|||||||:||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||MVPEPGPTANSTPAWGAGARXX-GGSGWVAAGKCVVIALTAAANSLLIALICTQPALRNTSNFFLVSLFTSDLMVGLVVMPPAMLNALYGRWVLARGLCLLWTAFDVMCCSASILNLCLI         {circumflex over ( )}10       {circumflex over( )}20        {circumflex over ( )}30       {circumflex over( )}40       {circumflex over ( )}50       {circumflex over( )}60       {circumflex over ( )}70        {circumflex over( )}80      {circumflex over ( )}90       {circumflex over( )}100      {circumflex over ( )}110         v130      v140      v150      v160      v170      v180      v190      v200      v210      v220      v230      v240SLDRYLLILSPLRYKLRMTPLRALALVLGAWSLAALASFLPLLLGWHELGHARPPVPGQCRLLASLPFVLVASGLTFFLPSGAICFTYCRILLAARKQAVQVASLTTGMASQASETLQVP|||||||||||||||||||||||||||||:||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||SLDRYLLILSPLRYKLRMTPLRALALVLGGWSLAALASFLPLLLGWHELGHARPPVPGQCRLLASLPFVLVASGLTFFLPSGAICFTYCRILLAARKQAVQVASLTTGMASQASETLQVP{circumflex over ( )}120      {circumflex over ( )}130      {circumflexover ( )}140      {circumflex over ( )}150      {circumflex over( )}160      {circumflex over ( )}170      {circumflex over( )}180      {circumflex over ( )}190      {circumflex over( )}200      {circumflex over ( )}210      {circumflex over( )}220      {circumflex over ( )}230         v250      v260      v270      v280      v290      v300      v310      v320      v330      v340      v350      v360RTPRPGVESADSRRLATKHSRKALKASLTLGILLGMFFVTWLPFFVANIVQAVCDCISPGLFDVLTWLGYCNSTMNPIIYPLFMRDFKRALGRFLPCPRCPRERQASLASPSLRTSHSGP|:| :||||||||||||||||:||||:||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||.::: :..   .  ::|RSPAAGVESADSRRLATKSSRKGLKASMTLGILLGMFFVTWLPFFVANIVQAVCDCISPGLFDVLTWLGYCNSTMNPIIYPLFMLDFKRALGRFLPCPRCPREPRPAWPRHHCAPLTAAP{circumflex over ( )}240      {circumflex over ( )}250      {circumflexover ( )}260      {circumflex over ( )}270      {circumflex over( )}280      {circumflex over ( )}290      {circumflex over( )}300      {circumflex over ( )}310      {circumflex over( )}320      {circumflex over ( )}330      {circumflex over( )}340      {circumflex over ( )}350         v370       v380       v390      v400      v410      v420      v430      v440RPGLSLQQVLPLP-LPPDSDSDSDAGSCC-SSGLRLTAQLLLPGEATQDPPLPTRAAAAVNFFNIDPAEPELRPHPLGIPTN |:|:  :  . |  ..:  ::::| ::  :.| | :  :| .   |.  | |.. :::::  ..||||||||||||||||GPALAYSRCCRCPCRRTQIRTQTQAQAAPRACGSRPSCCFLARPPRTPRCP-PGPLPPSISS-TSXPAEPELRPHPLGIPTN{circumflex over ( )}360      {circumflex over ( )}370      {circumflexover ( )}380      {circumflex over ( )}390      {circumflex over( )}400      {circumflex over ( )}410       {circumflex over( )}420       {circumflex over ( )}430

[0133]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 13 <210> SEQ ID NO 1<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 1gtcgaccctk tksgccmtca kcayrgrtcg cta 33 <210> SEQ ID NO 2 <211> LENGTH:35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer <400> SEQUENCE: 2 aagcttatga araagggcagscarcagagg kyrma 35 <210> SEQ ID NO 3 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer <400> SEQUENCE: 3 aagcatagca ggaaggcctt gaaggccagcctg 33 <210> SEQ ID NO 4 <211> LENGTH: 33 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:primer <400> SEQUENCE: 4 ggcgagaaat acgccctgaa gttctcccgg gac 33 <210>SEQ ID NO 5 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE:5 ttgccaatac tactctaagg tgcagcttcc 30 <210> SEQ ID NO 6 <211> LENGTH: 30<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: primer <400> SEQUENCE: 6 cacacgactt aactccatagagtcgatcgg 30 <210> SEQ ID NO 7 <211> LENGTH: 1914 <212> TYPE: DNA <213>ORGANISM: Rat <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(439)...(1749) <400> SEQUENCE: 7 ccaaccccca cgcgcgacac gtggtgatctaacgtactca cacgcccacc cttctcgaag 60 agactgcccc ggccggaagg cgggagttcggctcctgctc ccacatcccc agctgtgccc 120 ctagccagga accccacccc catcttatggcatccccggt ggccctattc catcccaggg 180 ctctcatcca gccccaagct aactttcattgactcgtcac atcagtaccc ctccccaaac 240 ttcttacccg agtactccag gtggccctgcgtaggaggca cccctacaac tcctcccgat 300 ctcttgaaat cgctgctcga tgacctaagaaccccgtttt gccaatacta ctctaaggtg 360 cagcttcctt tctcctcctt tgccttcaccctgtacctgc agtcaccata tcccgtcttg 420 gtcctcaacc cagtcccc atg gtt cca gagcca ggc cct gtc aac agt agc 471 Met Val Pro Glu Pro Gly Pro Val Asn SerSer 1 5 10 acc cca gcc tgg ggt ccc ggg cca ccg cct gct ccg ggg ggc agcggc 519 Thr Pro Ala Trp Gly Pro Gly Pro Pro Pro Ala Pro Gly Gly Ser Gly15 20 25 tgg gtg gct gcc gcg ctg tgc gtg gtc atc gtg ctg aca gca gcc gcc567 Trp Val Ala Ala Ala Leu Cys Val Val Ile Val Leu Thr Ala Ala Ala 3035 40 aat tcg ctg ctg atc gtg ctc att tgc acg cag ccc gcc gtg cgc aac615 Asn Ser Leu Leu Ile Val Leu Ile Cys Thr Gln Pro Ala Val Arg Asn 4550 55 acg tct aac ttc ttt ctg gtg tcg ctc ttc acg tcg gac ttg atg gtg663 Thr Ser Asn Phe Phe Leu Val Ser Leu Phe Thr Ser Asp Leu Met Val 6065 70 75 ggg ttg gtg gtg atg ccc cca gcc atg ctg aac gcg ctg tat ggg cgc711 Gly Leu Val Val Met Pro Pro Ala Met Leu Asn Ala Leu Tyr Gly Arg 8085 90 tgg gtg tta gct cga ggc ctc tgt ctg ctt tgg act gcc ttc gac gtg759 Trp Val Leu Ala Arg Gly Leu Cys Leu Leu Trp Thr Ala Phe Asp Val 95100 105 atg tgc tgc agc gcc tcc atc ctc aac ctc tgc ctc atc agc ctg gac807 Met Cys Cys Ser Ala Ser Ile Leu Asn Leu Cys Leu Ile Ser Leu Asp 110115 120 cgc tac ctg ctc atc ctc tcg ccg ctg cgc tac aag ctg cgc atg aca855 Arg Tyr Leu Leu Ile Leu Ser Pro Leu Arg Tyr Lys Leu Arg Met Thr 125130 135 gcc ccg cga gcc ctg gcg ctc atc ctg ggt gcc tgg agc ctc gcg gcg903 Ala Pro Arg Ala Leu Ala Leu Ile Leu Gly Ala Trp Ser Leu Ala Ala 140145 150 155 ctt gcc tcc ttc cta ccc ctc ttg ctg ggc tgg cac gaa ctg ggcaaa 951 Leu Ala Ser Phe Leu Pro Leu Leu Leu Gly Trp His Glu Leu Gly Lys160 165 170 gct cga aca cct gcc cct ggc cag tgc cgc cta ttg gcc agc ctgcct 999 Ala Arg Thr Pro Ala Pro Gly Gln Cys Arg Leu Leu Ala Ser Leu Pro175 180 185 ttt gtc ctc gtg gcg tcc ggc gtc acc ttt ttc ctg cct tcg ggtgcc 1047 Phe Val Leu Val Ala Ser Gly Val Thr Phe Phe Leu Pro Ser Gly Ala190 195 200 atc tgc ttc acc tac tgc agg atc ctt ctg gct gcc cgc aag caggcg 1095 Ile Cys Phe Thr Tyr Cys Arg Ile Leu Leu Ala Ala Arg Lys Gln Ala205 210 215 gtg caa gtg gcc tcg ctc acc acg ggc acg gct ggc cag gcc ttggaa 1143 Val Gln Val Ala Ser Leu Thr Thr Gly Thr Ala Gly Gln Ala Leu Glu220 225 230 235 acc ttg cag gtg ccc agg aca cca cgc cca ggg atg gag tccgct gac 1191 Thr Leu Gln Val Pro Arg Thr Pro Arg Pro Gly Met Glu Ser AlaAsp 240 245 250 agt agg cgt ctg gcc acc aag cat agc agg aag gcc ttg aaggcc agc 1239 Ser Arg Arg Leu Ala Thr Lys His Ser Arg Lys Ala Leu Lys AlaSer 255 260 265 ctg acc ctg ggc atc ctg ctg gga atg ttc ttt gtc acc tggctg ccc 1287 Leu Thr Leu Gly Ile Leu Leu Gly Met Phe Phe Val Thr Trp LeuPro 270 275 280 ttc ttt gtg gcc aac ata gct cag gcc gtg tgt gac tgc atctcc cca 1335 Phe Phe Val Ala Asn Ile Ala Gln Ala Val Cys Asp Cys Ile SerPro 285 290 295 ggc ctc ttc gat gtc ctc aca tgg ctg ggg tac tgt aat agcacc atg 1383 Gly Leu Phe Asp Val Leu Thr Trp Leu Gly Tyr Cys Asn Ser ThrMet 300 305 310 315 aac cct atc atc tac ccg ctc ttt atg cgg gac ttc aagagg gcc ctg 1431 Asn Pro Ile Ile Tyr Pro Leu Phe Met Arg Asp Phe Lys ArgAla Leu 320 325 330 ggc agg ttc ctg cat gcg tcc act gtc ccc cgg agc accggc cag ccc 1479 Gly Arg Phe Leu His Ala Ser Thr Val Pro Arg Ser Thr GlyGln Pro 335 340 345 tgc ctc ccc ctc cat gtg gac ctc tca cag cgg tgc cagacc agg cct 1527 Cys Leu Pro Leu His Val Asp Leu Ser Gln Arg Cys Gln ThrArg Pro 350 355 360 cag ctg cag cag gtg ctc gct ctg cct ctg ccg cca aactca gat tca 1575 Gln Leu Gln Gln Val Leu Ala Leu Pro Leu Pro Pro Asn SerAsp Ser 365 370 375 gac tcc gct tca ggg ggc acc tcg ggc ctg cag ctc acagcc cag ctt 1623 Asp Ser Ala Ser Gly Gly Thr Ser Gly Leu Gln Leu Thr AlaGln Leu 380 385 390 395 ctg ctg cct gga gag gcc aca cgg gac ccc ccg ccaccc acc agg gcc 1671 Leu Leu Pro Gly Glu Ala Thr Arg Asp Pro Pro Pro ProThr Arg Ala 400 405 410 acc act gtg gtc aac ttc ttt gtc aca gac tct gtggag cct gag ata 1719 Thr Thr Val Val Asn Phe Phe Val Thr Asp Ser Val GluPro Glu Ile 415 420 425 cgg ccg cat cca ctc agt tcc ccc gtg aactgaccaggtc aagagctggc 1769 Arg Pro His Pro Leu Ser Ser Pro Val Asn 430435 cattggaggc cacattcccg gagctctcag cccactctcc ctgagactag gaggtggtag1829 gtctcctgag agtgtgctga attgaggtat ctcagctagc ccatcttctg ctgcagctcc1889 ttgactgagg ggtagtcaga cacat 1914 <210> SEQ ID NO 8 <211> LENGTH:437 <212> TYPE: PRT <213> ORGANISM: Rat <400> SEQUENCE: 8 Met Val ProGlu Pro Gly Pro Val Asn Ser Ser Thr Pro Ala Trp Gly 1 5 10 15 Pro GlyPro Pro Pro Ala Pro Gly Gly Ser Gly Trp Val Ala Ala Ala 20 25 30 Leu CysVal Val Ile Val Leu Thr Ala Ala Ala Asn Ser Leu Leu Ile 35 40 45 Val LeuIle Cys Thr Gln Pro Ala Val Arg Asn Thr Ser Asn Phe Phe 50 55 60 Leu ValSer Leu Phe Thr Ser Asp Leu Met Val Gly Leu Val Val Met 65 70 75 80 ProPro Ala Met Leu Asn Ala Leu Tyr Gly Arg Trp Val Leu Ala Arg 85 90 95 GlyLeu Cys Leu Leu Trp Thr Ala Phe Asp Val Met Cys Cys Ser Ala 100 105 110Ser Ile Leu Asn Leu Cys Leu Ile Ser Leu Asp Arg Tyr Leu Leu Ile 115 120125 Leu Ser Pro Leu Arg Tyr Lys Leu Arg Met Thr Ala Pro Arg Ala Leu 130135 140 Ala Leu Ile Leu Gly Ala Trp Ser Leu Ala Ala Leu Ala Ser Phe Leu145 150 155 160 Pro Leu Leu Leu Gly Trp His Glu Leu Gly Lys Ala Arg ThrPro Ala 165 170 175 Pro Gly Gln Cys Arg Leu Leu Ala Ser Leu Pro Phe ValLeu Val Ala 180 185 190 Ser Gly Val Thr Phe Phe Leu Pro Ser Gly Ala IleCys Phe Thr Tyr 195 200 205 Cys Arg Ile Leu Leu Ala Ala Arg Lys Gln AlaVal Gln Val Ala Ser 210 215 220 Leu Thr Thr Gly Thr Ala Gly Gln Ala LeuGlu Thr Leu Gln Val Pro 225 230 235 240 Arg Thr Pro Arg Pro Gly Met GluSer Ala Asp Ser Arg Arg Leu Ala 245 250 255 Thr Lys His Ser Arg Lys AlaLeu Lys Ala Ser Leu Thr Leu Gly Ile 260 265 270 Leu Leu Gly Met Phe PheVal Thr Trp Leu Pro Phe Phe Val Ala Asn 275 280 285 Ile Ala Gln Ala ValCys Asp Cys Ile Ser Pro Gly Leu Phe Asp Val 290 295 300 Leu Thr Trp LeuGly Tyr Cys Asn Ser Thr Met Asn Pro Ile Ile Tyr 305 310 315 320 Pro LeuPhe Met Arg Asp Phe Lys Arg Ala Leu Gly Arg Phe Leu His 325 330 335 AlaSer Thr Val Pro Arg Ser Thr Gly Gln Pro Cys Leu Pro Leu His 340 345 350Val Asp Leu Ser Gln Arg Cys Gln Thr Arg Pro Gln Leu Gln Gln Val 355 360365 Leu Ala Leu Pro Leu Pro Pro Asn Ser Asp Ser Asp Ser Ala Ser Gly 370375 380 Gly Thr Ser Gly Leu Gln Leu Thr Ala Gln Leu Leu Leu Pro Gly Glu385 390 395 400 Ala Thr Arg Asp Pro Pro Pro Pro Thr Arg Ala Thr Thr ValVal Asn 405 410 415 Phe Phe Val Thr Asp Ser Val Glu Pro Glu Ile Arg ProHis Pro Leu 420 425 430 Ser Ser Pro Val Asn 435 <210> SEQ ID NO 9 <211>LENGTH: 2108 <212> TYPE: DNA <213> ORGANISM: Rat <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (439)...(1311) <221> NAME/KEY: intron<222> LOCATION: (1312)...(1505) <221> NAME/KEY: CDS <222> LOCATION:(1506)...(1943) <400> SEQUENCE: 9 ccaaccccca cgcgcgacac gtggtgatctaacgtactca cacgcccacc cttctcgaag 60 agactgcccc ggccggaagg cgggagttcggctcctgctc ccacatcccc agctgtgccc 120 ctagccagga accccacccc catcttatggcatccccggt ggccctattc catcccaggg 180 ctctcatcca gccccaagct aactttcattgactcgtcac atcagtaccc ctccccaaac 240 ttcttacccg agtactccag gtggccctgcgtaggaggca cccctacaac tcctcccgat 300 ctcttgaaat cgctgctcga tgacctaagaaccccgtttt gccaatacta ctctaaggtg 360 cagcttcctt tctcctcctt tgccttcaccctgtacctgc agtcaccata tcccgtcttg 420 gtcctcaacc cagtcccc atg gtt cca gagcca ggc cct gtc aac agt agc 471 Met Val Pro Glu Pro Gly Pro Val Asn SerSer 1 5 10 acc cca gcc tgg ggt ccc ggg cca ccg cct gct ccg ggg ggc agcggc 519 Thr Pro Ala Trp Gly Pro Gly Pro Pro Pro Ala Pro Gly Gly Ser Gly15 20 25 tgg gtg gct gcc gcg ctg tgc gtg gtc atc gtg ctg aca gca gcc gcc567 Trp Val Ala Ala Ala Leu Cys Val Val Ile Val Leu Thr Ala Ala Ala 3035 40 aat tcg ctg ctg atc gtg ctc att tgc acg cag ccc gcc gtg cgc aac615 Asn Ser Leu Leu Ile Val Leu Ile Cys Thr Gln Pro Ala Val Arg Asn 4550 55 acg tct aac ttc ttt ctg gtg tcg ctc ttc acg tcg gac ttg atg gtg663 Thr Ser Asn Phe Phe Leu Val Ser Leu Phe Thr Ser Asp Leu Met Val 6065 70 75 ggg ttg gtg gtg atg ccc cca gcc atg ctg aac gcg ctg tat ggg cgc711 Gly Leu Val Val Met Pro Pro Ala Met Leu Asn Ala Leu Tyr Gly Arg 8085 90 tgg gtg tta gct cga ggc ctc tgt ctg ctt tgg act gcc ttc gac gtg759 Trp Val Leu Ala Arg Gly Leu Cys Leu Leu Trp Thr Ala Phe Asp Val 95100 105 atg tgc tgc agc gcc tcc atc ctc aac ctc tgc ctc atc agc ctg gac807 Met Cys Cys Ser Ala Ser Ile Leu Asn Leu Cys Leu Ile Ser Leu Asp 110115 120 cgc tac ctg ctc atc ctc tcg ccg ctg cgc tac aag ctg cgc atg aca855 Arg Tyr Leu Leu Ile Leu Ser Pro Leu Arg Tyr Lys Leu Arg Met Thr 125130 135 gcc ccg cga gcc ctg gcg ctc atc ctg ggt gcc tgg agc ctc gcg gcg903 Ala Pro Arg Ala Leu Ala Leu Ile Leu Gly Ala Trp Ser Leu Ala Ala 140145 150 155 ctt gcc tcc ttc cta ccc ctc ttg ctg ggc tgg cac gaa ctg ggcaaa 951 Leu Ala Ser Phe Leu Pro Leu Leu Leu Gly Trp His Glu Leu Gly Lys160 165 170 gct cga aca cct gcc cct ggc cag tgc cgc cta ttg gcc agc ctgcct 999 Ala Arg Thr Pro Ala Pro Gly Gln Cys Arg Leu Leu Ala Ser Leu Pro175 180 185 ttt gtc ctc gtg gcg tcc ggc gtc acc ttt ttc ctg cct tcg ggtgcc 1047 Phe Val Leu Val Ala Ser Gly Val Thr Phe Phe Leu Pro Ser Gly Ala190 195 200 atc tgc ttc acc tac tgc agg atc ctt ctg gct gcc cgc aag caggcg 1095 Ile Cys Phe Thr Tyr Cys Arg Ile Leu Leu Ala Ala Arg Lys Gln Ala205 210 215 gtg caa gtg gcc tcg ctc acc acg ggc acg gct ggc cag gcc ttggaa 1143 Val Gln Val Ala Ser Leu Thr Thr Gly Thr Ala Gly Gln Ala Leu Glu220 225 230 235 acc ttg cag gtg ccc agg aca cca cgc cca ggg atg gag tccgct gac 1191 Thr Leu Gln Val Pro Arg Thr Pro Arg Pro Gly Met Glu Ser AlaAsp 240 245 250 agt agg cgt ctg gcc acc aag cat agc agg aag gcc ttg aaggcc agc 1239 Ser Arg Arg Leu Ala Thr Lys His Ser Arg Lys Ala Leu Lys AlaSer 255 260 265 ctg acc ctg ggc atc ctg ctg gga atg ttc ttt gtc acc tggctg ccc 1287 Leu Thr Leu Gly Ile Leu Leu Gly Met Phe Phe Val Thr Trp LeuPro 270 275 280 ttc ttt gtg gcc aac ata gct cag gtaaaatgat gaccgtgaaggtgggatgag 1341 Phe Phe Val Ala Asn Ile Ala Gln 285 290 cttaggtctgaccggagaga cgccatgctt cactgggcaa aggtgggagg gaggaggatg 1401 gctcatctgtggtgcctgtg tctgtgtttc tgtcctatcc ctgctgggtg ggtagcctgg 1461 gtccctgcctgggacatggg gtgtgatgag tcttatctcc acag gcc gtg tgt gac 1517 Ala Val CysAsp 295 tgc atc tcc cca ggc ctc ttc gat gtc ctc aca tgg ctg ggg tac tgt1565 Cys Ile Ser Pro Gly Leu Phe Asp Val Leu Thr Trp Leu Gly Tyr Cys 300305 310 aat agc acc atg aac cct atc atc tac ccg ctc ttt atg cgg gac ttc1613 Asn Ser Thr Met Asn Pro Ile Ile Tyr Pro Leu Phe Met Arg Asp Phe 315320 325 aag agg gcc ctg ggc agg ttc ctg cat gcg tcc act gtc ccc cgg agc1661 Lys Arg Ala Leu Gly Arg Phe Leu His Ala Ser Thr Val Pro Arg Ser 330335 340 acc ggc cag ccc tgc ctc ccc ctc cat gtg gac ctc tca cag cgg tgc1709 Thr Gly Gln Pro Cys Leu Pro Leu His Val Asp Leu Ser Gln Arg Cys 345350 355 cag acc agg cct cag ctg cag cag gtg ctc gct ctg cct ctg ccg cca1757 Gln Thr Arg Pro Gln Leu Gln Gln Val Leu Ala Leu Pro Leu Pro Pro 360365 370 375 aac tca gat tca gac tcc gct tca ggg ggc acc tcg ggc ctg cagctc 1805 Asn Ser Asp Ser Asp Ser Ala Ser Gly Gly Thr Ser Gly Leu Gln Leu380 385 390 aca gcc cag ctt ctg ctg cct gga gag gcc aca cgg gac ccc ccgcca 1853 Thr Ala Gln Leu Leu Leu Pro Gly Glu Ala Thr Arg Asp Pro Pro Pro395 400 405 ccc acc agg gcc acc act gtg gtc aac ttc ttt gtc aca gac tctgtg 1901 Pro Thr Arg Ala Thr Thr Val Val Asn Phe Phe Val Thr Asp Ser Val410 415 420 gag cct gag ata cgg ccg cat cca ctc agt tcc ccc gtg aac 1943Glu Pro Glu Ile Arg Pro His Pro Leu Ser Ser Pro Val Asn 425 430 435tgaccaggtc aagagctggc cattggaggc cacattcccg gagctctcag cccactctcc 2003ctgagactag gaggtggtag gtctcctgag agtgtgctga attgaggtat ctcagctagc 2063ccatcttctg ctgcagctcc ttgactgagg ggtagtcaga cacat 2108 <210> SEQ ID NO10 <211> LENGTH: 291 <212> TYPE: PRT <213> ORGANISM: Rat <400> SEQUENCE:10 Met Val Pro Glu Pro Gly Pro Val Asn Ser Ser Thr Pro Ala Trp Gly 1 510 15 Pro Gly Pro Pro Pro Ala Pro Gly Gly Ser Gly Trp Val Ala Ala Ala 2025 30 Leu Cys Val Val Ile Val Leu Thr Ala Ala Ala Asn Ser Leu Leu Ile 3540 45 Val Leu Ile Cys Thr Gln Pro Ala Val Arg Asn Thr Ser Asn Phe Phe 5055 60 Leu Val Ser Leu Phe Thr Ser Asp Leu Met Val Gly Leu Val Val Met 6570 75 80 Pro Pro Ala Met Leu Asn Ala Leu Tyr Gly Arg Trp Val Leu Ala Arg85 90 95 Gly Leu Cys Leu Leu Trp Thr Ala Phe Asp Val Met Cys Cys Ser Ala100 105 110 Ser Ile Leu Asn Leu Cys Leu Ile Ser Leu Asp Arg Tyr Leu LeuIle 115 120 125 Leu Ser Pro Leu Arg Tyr Lys Leu Arg Met Thr Ala Pro ArgAla Leu 130 135 140 Ala Leu Ile Leu Gly Ala Trp Ser Leu Ala Ala Leu AlaSer Phe Leu 145 150 155 160 Pro Leu Leu Leu Gly Trp His Glu Leu Gly LysAla Arg Thr Pro Ala 165 170 175 Pro Gly Gln Cys Arg Leu Leu Ala Ser LeuPro Phe Val Leu Val Ala 180 185 190 Ser Gly Val Thr Phe Phe Leu Pro SerGly Ala Ile Cys Phe Thr Tyr 195 200 205 Cys Arg Ile Leu Leu Ala Ala ArgLys Gln Ala Val Gln Val Ala Ser 210 215 220 Leu Thr Thr Gly Thr Ala GlyGln Ala Leu Glu Thr Leu Gln Val Pro 225 230 235 240 Arg Thr Pro Arg ProGly Met Glu Ser Ala Asp Ser Arg Arg Leu Ala 245 250 255 Thr Lys His SerArg Lys Ala Leu Lys Ala Ser Leu Thr Leu Gly Ile 260 265 270 Leu Leu GlyMet Phe Phe Val Thr Trp Leu Pro Phe Phe Val Ala Asn 275 280 285 Ile AlaGln 290 <210> SEQ ID NO 11 <211> LENGTH: 146 <212> TYPE: PRT <213>ORGANISM: Rat <400> SEQUENCE: 11 Ala Val Cys Asp Cys Ile Ser Pro Gly LeuPhe Asp Val Leu Thr Trp 1 5 10 15 Leu Gly Tyr Cys Asn Ser Thr Met AsnPro Ile Ile Tyr Pro Leu Phe 20 25 30 Met Arg Asp Phe Lys Arg Ala Leu GlyArg Phe Leu His Ala Ser Thr 35 40 45 Val Pro Arg Ser Thr Gly Gln Pro CysLeu Pro Leu His Val Asp Leu 50 55 60 Ser Gln Arg Cys Gln Thr Arg Pro GlnLeu Gln Gln Val Leu Ala Leu 65 70 75 80 Pro Leu Pro Pro Asn Ser Asp SerAsp Ser Ala Ser Gly Gly Thr Ser 85 90 95 Gly Leu Gln Leu Thr Ala Gln LeuLeu Leu Pro Gly Glu Ala Thr Arg 100 105 110 Asp Pro Pro Pro Pro Thr ArgAla Thr Thr Val Val Asn Phe Phe Val 115 120 125 Thr Asp Ser Val Glu ProGlu Ile Arg Pro His Pro Leu Ser Ser Pro 130 135 140 Val Asn 145 <210>SEQ ID NO 12 <211> LENGTH: 1647 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(135)...(1454) <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1647)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 12 actcacctcccccggggggc gtggtgagtc gcggtctgtt ctcacgacgg tccccgtcca 60 gcctgcggttcgccggggcc ctcatctgct ttcccgccac cctatcactc ccttgccgtc 120 caccctcggtcctc atg gtc cca gag ccg ggc cca acc gcc aat agc acc 170 Met Val Pro GluPro Gly Pro Thr Ala Asn Ser Thr 1 5 10 ccg gcc tgg ggg gca ggc gcc cgtcgn nng ggg ggc agc ggc tgg gtg 218 Pro Ala Trp Gly Ala Gly Ala Arg ArgXaa Gly Gly Ser Gly Trp Val 15 20 25 gcg gcc ggc ctg tgc gtg gtc atc gcgctg acg gcg gcg gcc aac tcg 266 Ala Ala Gly Leu Cys Val Val Ile Ala LeuThr Ala Ala Ala Asn Ser 30 35 40 ctg ctg atc gcg ctc atc tgc act cag cccgcg ctg cgc aac acg tcc 314 Leu Leu Ile Ala Leu Ile Cys Thr Gln Pro AlaLeu Arg Asn Thr Ser 45 50 55 60 aac ttc ttc ctg gtg tcg ctc ttc acg tctgac ctg atg gtc ggg ctg 362 Asn Phe Phe Leu Val Ser Leu Phe Thr Ser AspLeu Met Val Gly Leu 65 70 75 gtg gtg atg ccg ccg gcc atg ctg aac gcg ctgtac ggg cgc tgg gtg 410 Val Val Met Pro Pro Ala Met Leu Asn Ala Leu TyrGly Arg Trp Val 80 85 90 ctg gcg cgc ggc ctc tgc ctg ctc tgg acc gcc ttcgac gtg atg tgc 458 Leu Ala Arg Gly Leu Cys Leu Leu Trp Thr Ala Phe AspVal Met Cys 95 100 105 tgc agc gcc tcc atc ctc aac ctc tgc ctc atc agcctg gac cgc tac 506 Cys Ser Ala Ser Ile Leu Asn Leu Cys Leu Ile Ser LeuAsp Arg Tyr 110 115 120 ctg ctc atc ctc tcg ccg ctg cgc tac aag ctg cgcatg acg ccc ctg 554 Leu Leu Ile Leu Ser Pro Leu Arg Tyr Lys Leu Arg MetThr Pro Leu 125 130 135 140 cgt gcc ctg gcc cta gtc ctg ggc ggc tgg agcctc gcc gct ctc gcc 602 Arg Ala Leu Ala Leu Val Leu Gly Gly Trp Ser LeuAla Ala Leu Ala 145 150 155 tcc ttc ctg ccc ctg ctg ctg ggc tgg cac gagctg ggc cac gca cgg 650 Ser Phe Leu Pro Leu Leu Leu Gly Trp His Glu LeuGly His Ala Arg 160 165 170 cca ccc gtc cct ggc cag tgc cgc ctg ctg gccagc ctg cct ttt gtc 698 Pro Pro Val Pro Gly Gln Cys Arg Leu Leu Ala SerLeu Pro Phe Val 175 180 185 ctt gtg gcg tcg ggc ctc acc ttc ttc ctg ccctcg ggt gcc ata tgc 746 Leu Val Ala Ser Gly Leu Thr Phe Phe Leu Pro SerGly Ala Ile Cys 190 195 200 ttc acc tac tgc agg atc ctg cta gct gcc cgcaag cag gcc gtg cag 794 Phe Thr Tyr Cys Arg Ile Leu Leu Ala Ala Arg LysGln Ala Val Gln 205 210 215 220 gtg gcc tcc ctc acc acc ggc atg gcc agtcag gcc tcg gag acg ctg 842 Val Ala Ser Leu Thr Thr Gly Met Ala Ser GlnAla Ser Glu Thr Leu 225 230 235 cag gta ccc agg agc cca gcg gca ggg gtggag tct gct gac agc agg 890 Gln Val Pro Arg Ser Pro Ala Ala Gly Val GluSer Ala Asp Ser Arg 240 245 250 cgt cta gca acg aag agc agc agg aag ggcctg aag gcc agc atg acg 938 Arg Leu Ala Thr Lys Ser Ser Arg Lys Gly LeuLys Ala Ser Met Thr 255 260 265 ctg ggc atc ctg ctg ggc atg ttc ttt gtgacc tgg ttg ccc ttc ttt 986 Leu Gly Ile Leu Leu Gly Met Phe Phe Val ThrTrp Leu Pro Phe Phe 270 275 280 gtg gcc aac ata gtc cag gcc gtg tgc gactgc atc tcc cca ggc ctc 1034 Val Ala Asn Ile Val Gln Ala Val Cys Asp CysIle Ser Pro Gly Leu 285 290 295 300 ttc gat gtc ctc aca tgg ctg ggt tactgt aac agc acc atg aac ccc 1082 Phe Asp Val Leu Thr Trp Leu Gly Tyr CysAsn Ser Thr Met Asn Pro 305 310 315 atc atc tac cca ctc ttc atg ctg gacttc aag cgg gcg ctg ggc agg 1130 Ile Ile Tyr Pro Leu Phe Met Leu Asp PheLys Arg Ala Leu Gly Arg 320 325 330 ttc ctg cca tgt cca cgc tgt ccc cgggag ccc agg cca gcc tgg cct 1178 Phe Leu Pro Cys Pro Arg Cys Pro Arg GluPro Arg Pro Ala Trp Pro 335 340 345 cgc cat cac tgc gca cct ctc aca gcggcc ccc ggc ccg gcc tta gcc 1226 Arg His His Cys Ala Pro Leu Thr Ala AlaPro Gly Pro Ala Leu Ala 350 355 360 tac agc agg tgc tgc cgc tgc ccc tgccgc cgg act cag att cgg act 1274 Tyr Ser Arg Cys Cys Arg Cys Pro Cys ArgArg Thr Gln Ile Arg Thr 365 370 375 380 cag acg cag gct cag gcg gct cctcgg gcg tgc ggc tca cgg ccc agc 1322 Gln Thr Gln Ala Gln Ala Ala Pro ArgAla Cys Gly Ser Arg Pro Ser 385 390 395 tgc tgc ttc ctg gcg agg cca cccagg acc ccc cgc tgc cca cca ggg 1370 Cys Cys Phe Leu Ala Arg Pro Pro ArgThr Pro Arg Cys Pro Pro Gly 400 405 410 ccg ctg ccg ccg tca att tct tcaaca tcg sac ccc gcg gag ccc gag 1418 Pro Leu Pro Pro Ser Ile Ser Ser ThrSer Xaa Pro Ala Glu Pro Glu 415 420 425 ctg cgg ccg cat cca ctt ggc atcccc acg aac tga cccggcttgg 1464 Leu Arg Pro His Pro Leu Gly Ile Pro ThrAsn * 430 435 ggctggccaa tggggagctg gattgagcag aacccagacc ctgagtccttgggccagctc 1524 ttggctaaga ccaggaggct gcaagtctcc tagaagccct ctgagctccagaggggtgcg 1584 gcagagctga ccccctgctg ccatctccag gccccttacc tgcagggatcatagctgact 1644 aga 1647 <210> SEQ ID NO 13 <211> LENGTH: 439 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: (1)...(439) <223> OTHER INFORMATION: Xaa = AnyAmino Acid <400> SEQUENCE: 13 Met Val Pro Glu Pro Gly Pro Thr Ala AsnSer Thr Pro Ala Trp Gly 1 5 10 15 Ala Gly Ala Arg Xaa Xaa Gly Gly SerGly Trp Val Ala Ala Gly Leu 20 25 30 Cys Val Val Ile Ala Leu Thr Ala AlaAla Asn Ser Leu Leu Ile Ala 35 40 45 Leu Ile Cys Thr Gln Pro Ala Leu ArgAsn Thr Ser Asn Phe Phe Leu 50 55 60 Val Ser Leu Phe Thr Ser Asp Leu MetVal Gly Leu Val Val Met Pro 65 70 75 80 Pro Ala Met Leu Asn Ala Leu TyrGly Arg Trp Val Leu Ala Arg Gly 85 90 95 Leu Cys Leu Leu Trp Thr Ala PheAsp Val Met Cys Cys Ser Ala Ser 100 105 110 Ile Leu Asn Leu Cys Leu IleSer Leu Asp Arg Tyr Leu Leu Ile Leu 115 120 125 Ser Pro Leu Arg Tyr LysLeu Arg Met Thr Pro Leu Arg Ala Leu Ala 130 135 140 Leu Val Leu Gly GlyTrp Ser Leu Ala Ala Leu Ala Ser Phe Leu Pro 145 150 155 160 Leu Leu LeuGly Trp His Glu Leu Gly His Ala Arg Pro Pro Val Pro 165 170 175 Gly GlnCys Arg Leu Leu Ala Ser Leu Pro Phe Val Leu Val Ala Ser 180 185 190 GlyLeu Thr Phe Phe Leu Pro Ser Gly Ala Ile Cys Phe Thr Tyr Cys 195 200 205Arg Ile Leu Leu Ala Ala Arg Lys Gln Ala Val Gln Val Ala Ser Leu 210 215220 Thr Thr Gly Met Ala Ser Gln Ala Ser Glu Thr Leu Gln Val Pro Arg 225230 235 240 Ser Pro Ala Ala Gly Val Glu Ser Ala Asp Ser Arg Arg Leu AlaThr 245 250 255 Lys Ser Ser Arg Lys Gly Leu Lys Ala Ser Met Thr Leu GlyIle Leu 260 265 270 Leu Gly Met Phe Phe Val Thr Trp Leu Pro Phe Phe ValAla Asn Ile 275 280 285 Val Gln Ala Val Cys Asp Cys Ile Ser Pro Gly LeuPhe Asp Val Leu 290 295 300 Thr Trp Leu Gly Tyr Cys Asn Ser Thr Met AsnPro Ile Ile Tyr Pro 305 310 315 320 Leu Phe Met Leu Asp Phe Lys Arg AlaLeu Gly Arg Phe Leu Pro Cys 325 330 335 Pro Arg Cys Pro Arg Glu Pro ArgPro Ala Trp Pro Arg His His Cys 340 345 350 Ala Pro Leu Thr Ala Ala ProGly Pro Ala Leu Ala Tyr Ser Arg Cys 355 360 365 Cys Arg Cys Pro Cys ArgArg Thr Gln Ile Arg Thr Gln Thr Gln Ala 370 375 380 Gln Ala Ala Pro ArgAla Cys Gly Ser Arg Pro Ser Cys Cys Phe Leu 385 390 395 400 Ala Arg ProPro Arg Thr Pro Arg Cys Pro Pro Gly Pro Leu Pro Pro 405 410 415 Ser IleSer Ser Thr Ser Xaa Pro Ala Glu Pro Glu Leu Arg Pro His 420 425 430 ProLeu Gly Ile Pro Thr Asn 435

What is claimed is:
 1. Isolated human serotonin receptor protein St-B17comprising the amino acid sequence which encodes the third and sixthtransmembrane domains of SEQ ID NO:
 13. 2. A recombinant constructcomprising a polynucleotide encoding the human serotonin receptorprotein St-B17 of claim 1, operably linked to a heterologous promoter.3. The recombinant construct of claim 2, wherein said polynucleotidecomprises the nucleic acid sequence of SEQ ID NO:12 which encodes saidthird and and sixth transmembrane domains.
 4. An isolated polynucleotideencoding the human serotonin receptor protein St-B17 of claim
 1. 5. Theisolated polynucleotide of claim 4, wherein said polynucleotidecomprises the nucleic acid sequence of SEQ ID NO:12 which encodes saidthird and sixth transmembrane domains.
 6. A mammalian cell line incontinuous culture expressing the human serotonin receptor proteinSt-B17 of claim
 1. 7. The mammalian cell line of claim 6, wherein saidserotonin receptor protein St-B17 is encoded by a polynucleotidecomprising the nucleic acid sequence of SEQ ID NO:12 which encodes saidthird and sixth transmembrane domains.
 8. Isolated human serotoninreceptor protein St-B17 of claim 1 expressed by a polynucleotidecomprising the nucleic acid sequence of SEQ ID NO:12 which encodes saidthird and sixth transmembrane domains.
 9. Isolated rat serotoninreceptor protein St-B17 comprising the amino acid sequence which encodesthe third and sixth transmembrane domains of SEQ ID NO:8.
 10. Arecombinant construct comprising a polynucleotide encoding the ratserotonin receptor protein St-B17 of claim 9, operably linked to aheterologous promoter.
 11. The recombinant construct of claim 10,wherein said polynucleotide comprises the nucleic acid sequence of SEQID NO:7 which encodes said third and sixth transmembrane domains.
 12. Anisolated polynucleotide encoding the rat serotonin receptor proteinSt-B17 of claim
 9. 13. The isolated polynucleotide of claim 12, whereinsaid polynucleotide comprises the nucleic acid sequence of SEQ ID NO:7which encodes said third and sixth transmembrane domains.
 14. Amammalian cell line in continuous culture expressing the rat serotoninreceptor protein St-B17 of claim
 9. 15. The mammalian cell line of claim14, wherein said serotonin receptor protein St-B17 is encoded by apolynucleotide comprising the nucleic acid sequence of SEQ ID NO:7 whichencodes said third and sixth transmembrane domains.
 16. Isolated ratserotonin receptor protein St-B17 of claim 9 expressed by apolynucleotide comprising the nucleic acid sequence of SEQ ID NO:7 whichencodes said third and sixth transmembrane domains.